Chimeric nonhuman animal

ABSTRACT

The present invention relates to a method for producing a chimeric non-human animal expressing a desired protein, and a chimeric non-human animal or an offspring thereof expressing a desired protein. The present invention also relates to a method for analyzing the functions of a desired protein or a gene encoding the protein by comparing the phenotype of the above chimeric non-human animal with that of a corresponding wild-type animal.

TECHNICAL FIELD

The present invention relates to a method for producing a chimericnon-human animal expressing a desired protein, and a chimeric non-humananimal or an offspring thereof expressing a desired protein.

The present invention further relates to a method for analyzing thefunctions of a desired protein or a gene encoding such protein bycomparing the phenotype of the above chimeric non-human animal with thatof a corresponding wild-type animal.

BACKGROUND ART

The determination of the entire nucleotide sequence of the human genome(International Human Genome Sequencing Consortium, Nature, 409: 860-921,2001), the historic achievement of research, has provided at the sametime a new research theme for elucidating the functions of a largenumber of novel genes. Taking as an example human chromosome 22 (Dunhamet al., Nature, 402: 489-495, 1999), which is the second-smallestchromosome among the 24 human chromosomes, and whose entire nucleotidesequence was the first to be determined, the presence of a total of 545genes (pseudogenes excluded) has been inferred. The breakdown thereofis: 247 genes having known nucleotide 4 sequences and amino acidsequences; 150 novel genes showing homology with the known genes; and148 novel genes homologous to sequences with unknown functionsregistered with the Expressed Sequence Tag (EST) database. In addition,according to analyses made by software (GENESCAN) predicting genesdirectly from genomic sequences, 325 further novel genes whosetranscription products have remained unconfirmed have been predicted(Dunham et al., supra). Elucidation of in vivo functions of genes andthe products thereof (proteins) is not only important for understandingthe programs of life activities, but also can lead to development of newpharmaceuticals to overcome various human diseases. In other words,technological development for efficient functional analyses of novelgenes is a big issue in the fields of life science and medical researchin the post genome era.

As the most direct techniques to examine the in vivo functions ofmammalian genes, transgenic (Tg) mice wherein a foreign gene is insertedinto a mouse chromosome and the overexpression thereof is caused (Gordonet al., Proc. Natl. Acad. Sci. U.S.A., 77: 7380-7384, 1980), knock-out(KO) mice wherein an endogenous gene is disrupted (Shinichi Aizawa, BioManual Series 8, Gene Targeting, YODOSHA, 1995), and the like have beenwidely utilized.

Tg mice are generally produced by directly injecting purified DNAcontaining an expression unit comprising the cDNA of a gene to beexpressed (hereinafter referred to as a transgene), an appropriatepromoter, and a polyA addition site into a mouse fertilized egg nucleus(Gordon et al., supra). Since it has been shown that a Tg mouse having arat-derived growth hormone gene introduced therein grows to a sizenearly double that of a normal mouse (Palmiter et al., Nature, 300:611-615, 1982), Tg mice have been utilized to examine the in vivofunctions of numerous genes (and their products, proteins). A transgenein a Tg mouse may be expressed to a greater degree at a site differingfrom a site at which the gene is normally expressed by the action of apromoter or the like differing from the normal expression regulatorysystem. This may be generally referred to as ectopic overexpression. Aresponse at the whole body level that is induced by the ectopicoverexpression of a transgene can be said as highly importantinformation for inferring the in vivo functions of the protein.

The establishment of mouse embryonic stem cells (ES cells) havingpluripotency, the development of techniques to alter a target gene byhomologous recombination, and the like have been achieved since themiddle of the 1980's. Around 1990, the production of a mouse wherein aspecific gene was artificially disrupted (KO mouse) became possible(Capecchi, Trends Genet., 5: 70-76, 1989). For example, the fact thatthe homozygote of a KO mouse, wherein transcription factor GATA-2 thatis expressed in blood stem cells and vascular endothelia has beendisrupted, dies in the early developmental stage because of anemia hasindicated the importance of this transcription factor in hematopoiesis(Tsai et al., Nature, 371: 221-226, 1994). Many KO mice have beenproduced by many researchers to date. The analytical results haveprovided not only important information in a wide variety of fieldsranging from basic biology to clinical medicine, but also many humandisease model animals. Today, the use of KO mice is still the mostwidely used technique for elucidating the in vivo functions of a gene.

However, both the technique using Tg mice and the technique using KOmice require considerable time and effort, even for handling a singlegene. This problem has not been addressed to date. Thus, it has beenconsidered that the use of these techniques is unreaslistic for theexhaustive examination of the functions of many novel genes found fromthe above-mentioned genomic sequence information.

In a further simple method for elucidating the functions of a gene, avirus vector or a plasmid DNA containing the expression unit of a targetgene has been administered to an appropriate site in an animal, so as toexamine a phenotype induced by the following gene expression (Cannizzoet al., Nature Biotechnol., 15: 570-573, 1997; Ochiya et al., NatureMedicine, 5: 707-710, 1999). However under the current circumstances,such a method is not satisfactory as a technique for analyzing novelgenes with unknown functions, because it has many problems in terms ofintroduction efficiency, antigenicity, expression stability, and thelike.

Furthermore, when the functions of a humoral factor or a membraneprotein are analyzed, administration of a recombinant protein or aspecific antibody to an experimental animal is an effective means forfunctional analysis. However, preparing purified proteins or obtainingantibodies for various types of gene products with unknown functionscannot be said to be a realistic means, as in the cases of producing theabove Tg and KO mice.

To analyze the functions of secretory proteins such as hormones, growthfactors, and cytokines, or proteins prepared by altering membraneproteins to be secretory, an effective means involves artificiallyincreasing the effective concentration of such a protein in blood, orthe local effective concentration of the same, and then examining theeffect. As a method for increasing effective concentrations, theabove-described ectopic expression in Tg mice, administration of arecombinant protein, gene transfer into an individual, or the like hasbeen conventionally utilized. Above all, overexpression of a gene in Tgmice is the most widely employed method. This method has been utilizedfor examining the effect of the overexpression of a gene with knownfunctions (Palmiter et al., supra), or for identifying the functions ofa novel gene in few cases. Simonet et al. (Cell, 18: 309-319, 1997) haveproduced Tg mice expressing the cDNA encoding a novel gene with unknownfunctions under the regulation of ApoE gene promoter that is expressedspecifically in the liver to analyze the phenotype, thereby discoveringosteoprotegrin (OPG) exhibiting an effect of increasing bone quantitythrough the high level expression thereof.

However, a significant problem in the production of Tg mice is that atransgene is inserted randomly into a host chromosome, and theexpression is affected by the insertion position. For example, even whenthe promoter of a housekeeping gene showing constitutive strongexpression is utilized, an expected animal expressing the transgene athigh levels cannot be easily obtained. In general, it is thought thatthe first generation individuals (F0 individuals), which are born fromfertilized eggs containing foreign DNA injected therein, carry theintroduced DNA at an efficiency approximately ranging from 10% to 20%.Furthermore, it is thought that individuals showing expression asexpected account for approximately 0% to 20% of the DNA-introducedindividuals. Moreover, a problem that the number of copies of atransgene is unable to control may cause an event wherein the transgenesare inactivated because of the insertion of multiple copies thereof(Garrick et al., Nature Genet., 18: 56-59, 1998). Thus this is a reasoncausing a difficulty in obtaining individuals for expression.Furthermore, a requirement of maintaining and breeding many mice for along period to establish a Tg mouse strain expressing a transgene isalso a big problem. As described above, the probability of obtaining anexpected individual for expression in the production of Tg mice isgenerally several percent or less, so that there is a need to producemany F0 mice. Furthermore, it is inappropriate as described below todirectly use F0 mice, that have been confirmed to carry a transgene bythe analysis or the like of DNA derived from the tail tissue, forexpression analysis or phenotype analysis. Generally, detailed analysiscan be conducted only when F1 mice are obtained by crossing withwild-type mice.

It is considered possible to solve the above problems, the randominsertion into the host chromosome of the Tg mouse and theuncontrollable number of copies, by the use of gene targeting in mouseES cells (Shinichi Aizawa, supra; Capecchi, supra). Specifically, theeffect of insertion position on expression can be avoided by inserting(knock-in) a target gene downstream of an endogenous gene promoter, andthen causing the expression of the gene under the regulation of thepromoter. Knock-in has been conventionally utilized mainly for casessuch as those shown below.

(1) A foreign gene (e.g., marker gene) is inserted such that it issubstituted with a target gene. In this case, the target gene isdisrupted. Instead, the marker gene or the like is expressed withspecificity similar to that of the target gene. This is used for a case,for example, when a phenotype resulting from gene disruption isexamined, and the specificity of a promoter is examined at the sametime.

(2) The normal type of a target gene is substituted with a variant type(or an analogous gene) thereof. This technique is used for producing adisease model mouse by the expression of a variant of a specific gene,or for examining the interchangeability of gene functions.

As an early report concerning (1) above, Le Mouellic et al. (Proc. Natl.Acad. Sci. USA, 87: 4712-4716, 1990) have produced chimeric mice from EScells having the Escherichia coli LacZ gene inserted into the Hox-3.1gene locus, and showed that the LacZ gene product is expressed withspecificity similar to that of Hox-3.1. Moreover, Jin et al. (BBRC. 270:978-982, 2000) have inserted not only the LacZ gene in a manner similarto the above report, but also the Cre gene that has been ligated to aninternal ribosomal entry site (IRES; Jang et al., J. Virol., 62:2636-2643, 1988) downstream of the Lac Z gene into the Emx1 gene that isexpressed specifically in the cerebral cortex, thereby showing that theCre protein is expressed in the cerebral cortex.

In the meantime, there is a report as an example of (2) above that bythe insertion of a recombinant functional heavy chain VDJ variableregion gene fragment into a specific region of an antibody heavy chaingene, a mouse producing antibodies of all the classes having a commonvariable region has been produced. In a mouse wherein a VDJ fragmenthaving autoantigenic reactivity by itself, or having autoantigenicreactivity by a combination with a specific light chain has beeninserted upstream of a heavy chain constant region, B cells havedeveloped and differentiated normally, and antibody production closelyresembling physiological antibody production except for a single heavychain variable region being expressed has been observed (Sonoda et al.,Immunity, 6: 225-233, 1997).

As an example differing from both (1) and (2) above, there is a recentreport that a G418-resistance gene ligated to IRES has been insertedinto the downstream untranslated region (between the termination codonand the polyA addition site of the α1(I) procollagen (COL1A1) gene) ofthe α1(I) procollagen (COL1A1) gene of a sheep-foetus-derived fibroblast(McCreath et al., Nature, 405: 1066-1069, 2000). The sheep has beenproduced by transferring a cell nucleus derived from the abovefibroblast clone into the unfertilized egg of a sheep. In this case, theCOL1A1 gene is normally expressed, and G418-resistance gene is furtherexpressed with similar specificity.

As described above, knock-in is considered to be an effective techniqueto overcome the disadvantages of the method for producing Tg mice.However, there may still be some problems when the method is utilizedfor exhaustive gene analysis. First, the frequency of homologousrecombination in mouse ES cells is normally several percent or less.Considerable effort is required for obtaining only a homologousrecombinant clone wherein knock-in has been conducted downstream of anappropriate promoter for one type of a gene. Furthermore, in chimericmice produced from a knock-in ES cell line as described below, thechimerism (rate of the contribution of knock-in ES cells) differs inevery individual. In this case, the ratio of cell populations expressinga transgene to other cell populations varies depending on eachindividual and each tissue. This ratio may not be consistent withchimerism as determined from coat color, so that an understanding of theresults of phenotype analysis is accompanied by difficulties.Accordingly, utilization of mouse offspring, to which above knock-ingene locus is transmitted, is desired.

For both Tg and knock-in described above, it is generally desired toanalyze individuals after the transmission of a transgene to offsprings.However, in consideration of the experimental period, facilities, andeffort required for keeping mice, it is desired to be able to conductanalysis using the first generation (in the case of Tg, F0 individuals,and in the case of knock-in, chimeric individuals) for efficientanalyses of various types of genes. In the case of F0 individuals of Tgthat rely on random insertion, the use of the second generation, the F1individuals, is essential, because it is impossible to obtain againindividuals wherein transgenes have been inserted into the same site inthe same number of copies. On the other hand, in the case of knock-in,many chimeric individuals can be easily obtained from one type of ESclones. However, as described above, normally the chimerism differslargely depending on the individuals used, and individuals having a highchimerism that demonstrates the high level expression of a transgenecannot be easily obtained in large numbers. In addition, the chimerismis normally determined by coat color. However, the chimerism of tissueswhere a transgene is expressed may not always be consistent therewith.

In recent years, a system has been developed wherein all the cells of atissue of a chimeric mouse are derived from ES cells. This is referredto as blastocyst complementation (hereinafter described as the BCmethod). There have been successful examples of the BC method in T or Bcells of the immune system (Chen et al., Proc. Natl. Acad. Sci. U.S.A.,90: 4528-4532, 1993), lens (Liegeois et al., Proc. Natl. Acad. Sci.U.S.A., 93: 1303-1307, 1996), and the like. For example, the BC methodfor T or B cells has been developed to examine the functions of a genethat will be lethal when knock-out is conducted by a general method in Tor B cells. KO mice having a knocked-out RAG2 gene that is essential forsite-specific recombination taking place in early development of T or Bcells are unable to produce functional T or B cells. When a chimera isproduced using a RAG2-KO mouse embryo as a host with a wild-type EScell, T or B cells (derived from ES cells) nearly at the normal levelare observed in many chimeric mice regardless of the chimerism. That is,by the use of an ES cell wherein embryonic lethal genes in both alleleshave been knocked out (KO), only the functions of the gene in T or Bcells can be examined in a chimeric mouse.

The entire nucleotide sequence of the human genome has been determined.Desired now is a system allowing exhaustive in vivo functional analysesto be conducted on a large number of novel genes. For this purpose, itis required to be able to reliably, easily, and simultaneously producemany types of animals expressing transgenes at high levels. As describedabove, since conventional methods using such as Tg mice or knock-in areunable to satisfy this requirement, it has been thought that there is noconclusive method for conducting functional analyses of a large numberof genes obtained from genomic sequence information. Besides, it hasalso been considered difficult to directly utilize an individual forscreening for functions of a large number of genes. Among novel genesprovided by the human genome information, genes encoding secretoryproteins having homology with cytokines, growth factors, hormones andthe like can be said to be very interesting research objects in thatthey themselves can be pharmaceuticals. Hence, it is inferred thatdevelopment of new efficient techniques for analyzing the in vivofunctions of genes encoding secretory proteins and products thereof canlead to great advances in the development of pharmaceuticals fortreating human diseases.

As described above, mutant mice (knock-out/KO mice) that are producedfrom embryonic stem (ES) cells wherein a specific gene has beendisrupted by gene targeting are widely utilized as an essential tool inelucidation of the functions of genes (Shinichi Aizawa, supra).

Now, 10 or more years after KO mice were produced for the first time,the production of KO mice is recognized by many researchers as aprocedure that requires much effort and time. One major reason for thisis difficulty in obtaining homologous recombinants using ES cells. Aknock-out (KO) vector has a structure wherein, in a target gene genomicDNA fragment of a certain size (5 kb) or greater, a drug-resistancemarker is generally present after substitution with a target gene exonor insertion into the exon. By the electric pulse method employed ingeneral protocols, colonies that are resistant to the above drug areobtained at rates of 1 out of 10,000 to 1,000,000 ES cells used.However, most resistant clones are prepared by inserting an introductionvector randomly into a mouse chromosome. Thus, it is thought thathomologous recombination occurs in approximately 1 out of 100 to 10000such clones. To improve the rate of homologous recombinants to cloneswith random insertion, various studies have been conducted. For example,it has been reported that a longer length of the genomic DNA of ahomologous region contained in a vector is preferred (Deng & Capecchi,Mol. Cell. Biol., 12: 3365-71, 1992), or it has been reported that theuse of the genomic DNA (isogenic DNA) from a mouse strain that is thesame as that of a mouse ES cell to be subjected to targeting ispreferred (Deng & Capecchi, supra). Furthermore, in the method that iscurrently most widely used, a KO vector containing a negative selectionmarker outside the genomic DNA of a homologous region is used inaddition to the above selection marker. This method utilizes an eventwhereby a toxic negative marker is expressed in a clone prepared byrandom insertion so as to cause the cell to die; however, this does notoccur in a homologous recombinant. As negative selection markers, theHSV-tk gene (a thymidine analog such as ganciclovir, FIAU, or the likeis required to be added to a medium) reported by Mansour et al. (Nature,336: 348-352, 1988), DT-A (diphtheria toxin A chain) reported by Yagi etal. (Anal. Biochem., 214: 77-86, 1993), and the like are known. Whenthis negative selection method functions as theorized, a resultpredicted is that all colonies would be homologous recombinants. Howeverin actual cases, the results vary widely among reports, and theresulting enrichment effect is thought to be, in general, several timesgreater than those of other cases. Even under current actual situationswhere the negative selection method is generally employed in knock-outexperiments, obtaining 100 to 1000 clones with random insertion andselecting homologous recombinants from these clones are carried outregularly.

In addition to the production of mice from variant ES cells, mammaliancell lines prepared by the alteration or disruption of genes byhomologous recombination are themselves important materials forelucidating the functions of the genes. Furthermore, homologousrecombination has been considered to be an ultimate therapy for diseases(hereditary diseases) caused by gene deletion or mutation. However, theratio of homologous recombinants to clones with random insertion inmammalian cell lines or primary cultured cells is equivalent to, orlower than that in mouse ES. In application for the analysis of genefunctions or gene therapy at the cellular level, improvement in theabove ratio has been expected (Yanez & Poter, Gene Therapy, 5: 149-159,1998). As attempts other than the above cases concerning improvement inthe ratio of homologous recombinants to clones with random insertion,(1) high level expression, inactivation, inhibition, and the like ofproteins (genes) involved in DNA recombination or repair, (2) specificdamages to target genes, and the like have been examined. However, nomethod that can provide drastic improvement in the ratio of homologousrecombinants in a chromosome gene, is simple, and has highreproducibility has been known (Yanez & Poter, supra; Vasquez et al.,Proc. Natl. Acad. Sci. U.S.A., 98: 8403-8410, 2001).

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a simple and highlyreproducible method for analyzing gene functions.

In the present invention, by the utilization of gene targeting byhomologous recombination, a desired gene is inserted into a gene locusfor the expression in any cells and/or tissues, for example, a mouse Igκgene locus, in a chimeric non-human animal. Gene targeting meansaltering a specific gene in a chromosome utilizing homologousrecombination. Efficiently obtaining cells wherein homologousrecombination has occurred is also important as an object to beachieved.

As a result of intensive studies to achieve the above objects, thepresent inventors have produced a chimeric mouse from a mouse ES cellwherein a structural gene encoding a secretory protein is inserteddownstream of an immunoglobulin light chain gene, and a host embryoderived from a B-lymphocyte-deficient strain, and then found that in theB cell of the chimeric mouse, the above secretory protein is expressedat high levels. Furthermore, the present inventors have found a simplemethod for improving the ratio of homologous recombinants to clones withrandom insertion of a targeting vector in a mammalian cell, and thushave completed the present invention.

In a chimeric animal produced by the method of the present invention, anoverexpression effect of the product derived from the introducedstructural gene is observed regardless of the chimerism based on coatcolor. By the use of this system, a chimeric animal expressing atransgene more efficiently and more reliably at high levels than ispossible with conventional methods can be obtained. This high efficiencywhen compared with that of conventional methods is based mainly on thefact that by the use of the B-lymphocyte-deficient host embryo, all theB lymphocytes of the chimeric animal are derived from the ES cells,regardless of the chimerism. In particular, the high efficiency obtainedwhen the regulatory region of an immunoglobulin light chain gene isutilized is based on the following points:

-   -   (1) Homologous recombination occurs in Igκ gene locus at an        efficiency of 5% or more.    -   (2) The expression system of immunoglobulin is utilized.    -   (3) The expression of immunoglobulin is very low in the early        developmental stage, and shows an explosive increase at and        after the weanling stage. Thus, even when a gene whose high        level expression causes embryonic lethality is introduced, the        functions of the gene in an adult can be examined.

The present invention is summarized as follows.

In a first aspect, the present invention provides a method for producinga chimeric non-human animal, which comprises the steps of:

-   -   1) preparing a pluripotent cell derived from a non-human animal        containing a genome wherein a nucleic acid sequence encoding a        desired protein is located so that the expression of the desired        protein is regulated by the regulatory region of a gene that is        expressed in at least a specific cell and/or tissue;    -   2) obtaining a chimeric embryo by injecting the pluripotent cell        prepared in the above step 1 into a host embryo of a non-human        animal strain that is deficient in the above specific cell        and/or tissue;    -   3) transplanting the chimeric embryo obtained in the above step        2 to a foster mother of non-human animal of the same species;        and    -   4) selecting a chimeric non-human animal expressing the desired        protein in at least the above specific cell and/or tissue from        offsprings obtained after the above transplantation step 3.

In one embodiment of the present invention, the above nucleic acidsequence encoding a desired protein is located downstream of theregulatory region of a gene that is expressed in a specific cell and/ortissue. In another embodiment, the above nucleic acid sequence encodinga desired protein is located downstream of an internal ribosomal entrysite located downstream of the termination codon of a gene that isexpressed in a specific cell and/or tissue.

In still another embodiment, a sequence containing an internal ribosomalentry site and the above nucleic acid sequence encoding a desiredprotein is located between the termination codon and a polyA signalregion of a gene that is expressed in a specific cell and/or tissue.

In still another embodiment, a sequence containing a polyA signalregion, a promoter sequence, and the above nucleic acid sequenceencoding a desired protein is located between the termination codon anda polyA signal region of a gene that is expressed in a specific celland/or tissue. Here, the above promoter sequence is preferably derivedfrom a gene that is expressed in a specific cell and/or tissue.Furthermore, the polyA signal region to be located together with theabove nucleic acid sequence encoding a desired protein may be same as ordifferent from the polyA signal region of a gene that is expressed in aspecific cell and/or tissue.

In still another embodiment, a sequence containing a promoter sequence,the above nucleic acid sequence encoding a desired protein, and a polyAsignal region is located downstream of a polyA signal region of a genethat is expressed in a specific cell and/or tissue. Here, the abovepromoter sequence is preferably derived from a gene that is expressed ina specific cell and/or tissue. Furthermore, the polyA signal region tobe located together with the above nucleic acid sequence encoding adesired protein may be the same as or different from the polyA signalregion of a gene that is expressed in a specific cell and/or tissue.Furthermore, the distance between the polyA signal region of the genethat is expressed in a specific cell and/or tissue and the abovepromoter sequence is preferably less than 1 Kb.

In still another embodiment of the present invention, the pluripotentcell contains both a genome wherein the nucleic acid sequence encoding adesired protein is located so that the expression of the desired proteinis regulated by the regulatory region of a gene that is expressed in atleast a specific cell and/or tissue, and a genome wherein the allele ofthe gene that is expressed in the above specific cell and/or tissue isinactivated.

In another embodiment of the present invention, the pluripotent cell isan embryonic stem cell.

In still another embodiment of the present invention, the chimericnon-human animal is selected from the group consisting of mice, cattle,pigs, monkeys, rats, sheep, goats, rabbits, and hamsters. In a preferredembodiment of the present invention, the chimeric non-human animal is amouse.

In still another embodiment of the present invention, a combination of agene that is expressed in a specific cell and/or tissue and the specificcell and/or tissue deficient in a non-human animal strain is selectedfrom the group consisting of the following (1) to (7):

-   (1) an immunoglobulin light chain or heavy chain gene and a    B-lymphocyte;-   (2) a T-cell receptor gene and a T-lymphocyte;-   (3) a myoglobin gene and a muscle cell;-   (4) a crystallin gene and a crystalline lens of an eyeball;-   (5) a renin gene and a kidney tissue;-   (6) an albumin gene and a liver tissue; and-   (7) a lipase gene and a pancreas tissue.

In a preferred embodiment of the present invention, the combination of agene that is expressed in a specific cell and/or tissue and the specificcell and/or tissue deficient in a non-human animal strain is that of animmunoglobulin light chain K gene and a B-lymphocyte.

In a second aspect, the present invention provides a method forproducing a non-human animal expressing a desired protein, comprisingobtaining an offspring capable of expressing the desired protein bycrossing a chimeric non-human animal produced by the above method forproducing a chimeric non-human animal.

Furthermore, in a third aspect, the present invention provides achimeric non-human animal, which is derived from a pluripotent cellderived from a non-human animal containing a genome wherein a nucleicacid sequence encoding a desired protein is located so that theexpression of the desired protein is regulated by the regulatory regionof a gene that is expressed in a specific cell and/or tissue and a hostembryo of a non-human animal strain deficient in the above specific celland/or tissue, and is capable of expressing the above desired protein inat least the above specific cells and/or tissues.

In one embodiment of the present invention, the above nucleic acidsequence encoding a desired protein is located downstream of theregulatory region of a gene that is expressed in a specific cell and/ortissue. In another embodiment, the above nucleic acid sequence encodinga desired protein is located downstream of an internal ribosomal entrysite located downstream of the termination codon of a gene that isexpressed in a specific cell and/or tissue.

In still another embodiment, a sequence containing an internal ribosomalentry site and the above nucleic acid sequence encoding a desiredprotein is located between the termination codon and a polyA signalregion of a gene that is expressed in a specific cell and/or tissue.

In still another embodiment, a sequence containing a polyA signalregion, a promoter sequence, and the above nucleic acid sequenceencoding a desired protein is located between the termination codon anda polyA signal region of a gene that is expressed in a specific celland/or tissue. Here, the above promoter sequence is preferably derivedfrom a gene that is expressed in a specific cell and/or tissue.Furthermore, the polyA signal region to be located together with theabove nucleic acid sequence encoding a desired protein may be same as ordifferent from the polyA signal region of a gene that is expressed in aspecific cell and/or tissue.

In still another embodiment, a sequence containing a promoter sequence,the above nucleic acid sequence encoding a desired protein, and a polyAsignal region is located downstream of a polyA signal region of a genethat is expressed in a specific cell and/or tissue. Here, the abovepromoter sequence is preferably derived from a gene that is expressed ina specific cell and/or tissue. Furthermore, the polyA signal region tobe located together with the above nucleic acid sequence encoding adesired protein may be the same as or different from the polyA region ofa gene that is expressed in a specific cell and/or tissue. Furthermore,the distance between the polyA signal region of a gene that is expressedin a specific cell and/or tissue and the above promoter sequence ispreferably less than 1 Kb.

In still another embodiment of the present invention, the pluripotentcell contains both a genome wherein the nucleic acid sequence encoding adesired protein is located so that the expression of the desired proteinis regulated by the regulatory region of a gene that is expressed in atleast a specific cell and/or tissue, and a genome wherein the allele ofthe gene that is expressed in the above specific cell and/or tissue isinactivated.

In another embodiment of the present invention, the pluripotent cell isan embryonic stem cell.

In still another embodiment of the present invention, the chimericnon-human animal is selected from the group consisting of mice, cattle,pigs, monkeys, rats, sheep, goats, rabbits, and hamsters. In a preferredembodiment of the present invention, the animal is a mouse.

In still another embodiment of the present invention, a combination of agene that is expressed in a specific cell and/or tissue and the specificcell and/or tissue deficient in a non-human animal strain is selectedfrom the group consisting of the following (1) to (7):

-   (1) an immunoglobulin light chain or heavy chain gene and a    B-lymphocyte;-   (2) a T-cell receptor gene and a T-lymphocyte;-   (3) a myoglobin gene and a muscle cell;-   (4) a crystallin gene and a crystalline lens of an eyeball;-   (5) a renin gene and a kidney tissue;-   (6) an albumin gene and a liver tissue; and-   (7) a lipase gene and a pancreas tissue.

In a preferred embodiment of the present invention, the combination of agene that is expressed in a specific cell and/or tissue and the specificcell and/or tissue deficient in a non-human animal strain is that of animmunoglobulin light chain K gene and a B-lymphocyte.

Furthermore, in a fourth aspect, the present invention provides a methodfor analyzing the in vivo functions of a desired protein or a geneencoding the desired protein, comprising comparing the phenotype of theabove chimeric non-human animal or an offspring of the above chimericnon-human animal capable of expressing the desired protein with that ofa corresponding wild-type non-human animal containing no nucleic acidsequence of a gene encoding the desired protein, so as to determinedifferences in these phenotypes.

Furthermore, in a fifth aspect, the present invention provides a tissueor cell derived from any of the above chimeric non-human animals. Thecell or tissue contains a genome wherein a nucleic acid sequenceencoding a desired protein is located so that the expression of thedesired protein is regulated by the regulatory region of a gene that isexpressed in the cell or the tissue and can express the desired protein.

In one embodiment of the present invention, the above tissue or cell isselected from the group consisting of B-lymphocytes, T-lymphocytes,muscle cells, crystalline lenses of eyeballs, kidney tissues, livertissues, and pancreas tissues.

In a sixth aspect, the present invention also provides a hybridoma thatis produced by the fusion of a cell derived from the above tissue orcell and a proliferable tumor cell.

Furthermore, in a seventh aspect, the present invention provides amethod for producing a desired protein, comprising causing theproduction of the desired protein using any of the above chimericnon-human animals, the above tissues or cells, or the above hybridomas,and then collecting the proteins.

The terms relating to the present invention are as defined below.

The term “non-human animal” used in this specification means an animalexcluding humans, and is generally selected from vertebrates comprisingfish, reptiles, amphibians, birds, and mammals, and is preferablyselected from mammals. In the production of chimeric non-human animalsin the present invention, embryonic stem cells are preferably utilizedas pluripotent cells. Thus, the non-human animals intended by thepresent invention encompass all non-human animals including non-humananimals for which embryonic stem cells can be established (e.g., mice,cattle, sheep, pigs, hamsters, monkeys, goats, rabbits, and rats), andother non-human animals for which embryonic stem cells can beestablished in the future. The term “chimeric non-human animal” means ananimal formed of both differentiated cells derived from a pluripotentcell (described later), and differentiated cells derived from a hostembryo (Bradley et al., Nature, 309: 255-6, 1984). Experimentally, thebirth of an animal (0% chimera) wherein all the cells are derived from ahost embryo, or an animal (100% chimera) wherein all the cells arederived from a pluripotent cell is possible. Strictly speaking, theseanimals are not “chimera.” However, the term “chimeric non-human animal”encompasses these animals for convenience.

The term “pluripotent cell” or “cell having pluripotency” used in thisspecification means, in the production of the above chimeric non-humananimal, a cell that can differentiate into 2 or more types of cells ortissues of a chimeric non-human animal by injection into a host embryoor by formation of aggregate embryos. Specific examples of suchpluripotent cells include embryonic stem cells (ES cells), embryonicgerm cells (EG cells), and embryonal carcinoma cells (EC cells).

The term “embryonic stem cell” used in this specification is alsoreferred to as an ES cell (Embryonic Stem cell), and means an earlyembryo-derived cultured cells characterized in that such cells canproliferate while maintaining anaplasticity (totipotency). Specifically,embryonic stem cells are of a cell line that is established by culturingcells of an inner cell mass that are undifferentiated stem cells,existing inside the blastocyst in an early embryo of an animal, so thatthe cells keep proliferating while maintaining their undifferentiatedstate. Furthermore, the term “embryonic germ cell” used in thisspecification is also referred to as an EG cell, and means a culturedcell derived from a primordial germ cell, which is characterized in thatit has ability almost equivalent to that of the above embryonic stemcell. The embryonic germ cells are of a cell line that is established byculturing primordial germ cells obtained from an embryo several days toseveral weeks after fertilization (for example, in the case of a mouse,an approximately 8.5 days old embryo) so that the cells keepproliferating while maintaining their undifferentiated state.

The term “desired protein” used in this specification refers to aprotein to be expressed is attempted in at least 1 type of cell and/ortissue of a chimeric non-human animal produced by the method of thepresent invention. The functions of such a protein may be either knownor unknown. The above desired protein may be a functional secretoryprotein, a functional membrane protein, a functional intracellular orintranuclear protein, a soluble portion of a functional membrane proteinhaving a secretory signal added thereto, or the like, which are derivedfrom mammals. Here, the term “functional” means to have a specific typeof work, action, or function in vivo.

In the case of a protein with known functions, new findings may beobtained about the interactive relationship of the functions of theprotein by observing the effect of high level expression of the desiredprotein in at least one type of cell and/or tissue of a chimericnon-human animal. In the case of a protein with unknown functions,findings leading to the elucidation of the functions of the protein mayalso be obtained by observing the effect of the high level expression.The “desired protein” in the present invention will be expressed in achimeric non-human animal into which the gene thereof has beenintroduced. The “desired protein” may be not expressed or is expressedpoorly in a specific cell and/or tissue that is caused to express theprotein according to the present invention, or it may refer to a proteinderived from an heterologous animal. The types of the desired proteinmay be any proteins of interest.

The term “nucleic acid sequence encoding a desired protein” used in thisspecification may refer to either endogenous or exogenous DNA. Examplesof the exogenous DNA include a human-derived DNA. In this specification,the term “a structural gene encoding a desired protein” is used as asynonym for the above term.

The term “expression” used with reference to a protein in thisspecification has a meaning equivalent to the same term used withreference to the expression of a gene encoding the protein.

The term “regulatory region” used in this specification refers to ageneral term such as “regulatory sequence,” “control sequence,” “controlregion,” or the like, and indicates a region having functions toregulate or control gene expression (transcription, translation, orprotein synthesis). Examples of such a regulatory region include, butare not limited to, a promoter, an enhancer, and a silencer.Furthermore, the term “regulatory region” in the present invention mayrefer to a region containing one functional element (e.g., one promotersequence) or a region containing multiple elements (e.g., 1 promotersequence, 1 enhancer sequence, and the like). Furthermore, the term“promoter sequence” refers to one type of a regulatory region known bypersons skilled in the art, and indicates a nucleotide sequence that islocated upstream of a structural gene to which RNA polymerase binds uponthe start of transcription.

The term “internal ribosomal entry site” used in this specification isabbreviated as IRES (Internal Ribosomal Entry Site), and is known as anelement that enables polycistronic expression. An IRES indicates a sitethat forms a unique RNA secondary structure, and enables the initiationof translation by a ribosome from the initiation codon locateddownstream of the RNA secondary structure. In the case of mammals, anIRES is inferred to be involved in the event of the initiation oftranslation and protein synthesis by binding to the subunit for thedecoding of a ribosome so as to cause a conformational change whereby anadjacent region encoding a protein is brought into the decoding site(Spahn et al., Science 291: 1959, 2001).

The term “polyA signal region” used in this specification indicates anucleotide sequence portion that is located at the end portion of atranscription region and directs the addition of polyadenylic acid (A)chain to the 3′ untranslated region of an mRNA precursor aftertranscription.

The terms “upstream” and “downstream” used in this specificationindicates directions toward the 5′ end and the 3′ end, respectively, ina nucleic acid sequence such as a genome and a polynucleotide.

The terms “bp (base pair)” and “Kb (kilobase pair)” used in thisspecification indicate the length and distance of a nucleic acidsequence. 1 bp represents one base pair, and 1 Kb corresponds to 1000bp.

The term “allele” used in this specification means a gene that islocated in homologous regions of a homologous chromosome, and is alsofunctionally homologous in an organism having a genome that ispolyploid. Both alleles are generally expressed. Furthermore, the term“allelic exclusion” in terms of an individual organism refers to asituation where, although a trait derived from both alleles isrepresented, only either one of the alleles is randomly expressed inindividual cells, and the expression of the other is excluded. Forexample, this is found in the gene of an antibody or a T cell receptor,and is due to the fact that only one complete gene is formed by therecombination of one variable region gene as a signal.

The term “soluble portion of a membrane protein having a secretorysignal added thereto” used in this specification means an extracellulardomain among membrane protein molecules, to which a secretory signal (ora signal sequence) has bound.

The term “immunoglobulin light chain gene” used in this specificationindicates a gene encoding the light chain (or L chain) of animmunoglobulin (Ig) molecule. The light chain includes the κ chain andthe λ chain, and is composed of a variable (V) region and a constant (C)region. Moreover, a light chain gene is composed of one C region gene,multiple V region genes, and multiple junction (J) region genes.

The term “host embryo of a non-human animal strain deficient in aspecific cell and/or tissue” or “deficient host embryo” used in thisspecification refers to a host non-human animal embryo for injecting apluripotent cell, and means an embryo deficient in the cell and/ortissue.

The term “offspring” used with reference to the chimeric non-humananimal in this specification refers to an offspring obtained by thecrossing of chimeric non-human animals of the present invention, or achimeric non-human animal of the present invention with a non-humananimal of the same species, and means a non-human animal expressing adesired protein in at least a specific cell and/or tissue.

The term “phenotype” used in this specification indicates an originaltrait of an animal, or a trait of an animal that appears as a result ofthe presence a transgene.

The term “tumor cell capable of proliferation” used in thisspecification means a cell capable of permanently proliferating bytumorigenesis. Examples of such a tumor cell include cells ofplasmocytoma (e.g., myeloma cells) that produce immunoglobulins.

The term “hybridoma” used in this specification indicates a hybrid cellthat is formed by fusing a cell derived from a tissue or a cell obtainedfrom the chimeric non-human animal of the present invention or anoffspring thereof with the above tumor cell capable of proliferation.

The term “knock-in vector” used in this specification indicates a vectorthat is used for the expression of a desired protein by introducing agene encoding the desired protein to a target gene locus by homologousrecombination. In addition, the term “knock-in” or “gene knock-in”refers to the introduction of a nucleic acid sequence encoding a desiredprotein to a target gene locus by homologous recombination, therebycausing the expression of the desired protein.

The term “knock-out vector” used in this specification indicates avector for disrupting or inactivating a target gene of a non-humananimal by homologous recombination. Moreover, the term “knock-out” or“gene knock-out” means the introduction of a structure that inhibits theexpression of a gene to the target gene locus by homologousrecombination, thereby disrupting or inactivating the target gene.

The term “targeting vector” used in this specification indicates avector that can be introduced by homologous recombination to a targetposition in the genome of a non-human animal. The term “targetingvector” encompasses the above “knock-in vector” and “knock-out vector.”In addition, the term “targeting” or “gene targeting” means to introduceby homologous recombination a desired gene structure to a targetposition in the genome of a non-human animal.

The term “polyamine” used in this specification is a general termreferring to a linear aliphatic hydrocarbon having 2 or more primaryamino groups.

The present invention will be described in detail as follows. Thisapplication claims a priority from Japanese Patent Application No.2002-42321 filed Feb. 19, 2002, which claims a priority from JapanesePatent Application No. 2001-350481 filed Nov. 15, 2001. This applicationincludes content disclosed in the specifications and/or drawings of theabove Japanese Patent Applications.

The present inventors have developed a method for producing a chimericnon-human animal expressing a desired protein at high levels in at leasta specific cell and/or tissue without being largely affected bychimerism by applying the above BC method. That is, the method forproducing a chimeric non-human animal of the present invention comprisesthe steps of:

-   -   1) preparing a pluripotent cell derived from a non-human animal        that contains a genome wherein a nucleic acid sequence encoding        a desired protein is located so that the expression of the        desired protein is regulated by the regulatory region of a gene        that is expressed in at least a specific cell and/or tissue;    -   2) obtaining a chimeric embryo by injecting the pluripotent cell        prepared in the above step 1 into a host embryo of a non-human        animal strain deficient in the above specific cell and/or        tissue;    -   3) transplanting the chimeric embryo obtained in the above step        2 into a foster parent non-human animal of the same species; and    -   4) selecting a chimeric non-human animal expressing the desired        protein in at least the above specific cell and/or tissue from        among offsprings obtained after the transplantation in the above        step 3.

In the method of the present invention, the deficient cell and/or tissuein a host embryo is complemented by the above pluripotent cell byblastocyst complementation (BC) as described above. Specifically,regardless of the chimerism of the produced whole chimeric non-humananimal, all the cells and/or tissues are derived from the pluripotentcell. As a result, as long as the pluripotent cell carries a gene(nucleic acid sequence) encoding a desired protein so that the gene isregulated by the regulatory sequence of a gene that is expressed in theabove cell and/or tissue, high level expression of the desired proteinin the cell and/or tissue of a chimeric non-human animal can beexpected.

1. Preparation of Pluripotent Cells

In the method for producing a chimeric non-human animal of the presentinvention, first, a pluripotent cell derived from a non-human animal isprepared, such cell containing a genome wherein a nucleic acid sequence(structural gene) encoding a desired protein is located (inserted). Thenucleic acid sequence is located so that the expression of the desiredprotein is regulated by the regulatory region of a gene that isexpressed in a specific cell and/or tissue.

As the cell having pluripotency in the present invention, those asdefined above can be utilized. Embryonic stem cells (ES cells) arepreferred, and particularly, mouse ES cells are preferred.

In addition, a gene that is expressed in a specific cell and/or tissuemay be expressed tissue-specifically or constitutively. Examples of agene that is expressed tissue-specifically include an immunoglobulinlight chain or heavy chain gene, a T cell receptor gene, a myoglobingene, a crystallin gene, a renin gene, a lipase gene, and an albumingene. Furthermore, an example of a gene that is constitutively expressedis a hypoxanthine guanine phosphoribosyl transferase (HPRT) gene. Whenthe gene is a gene that is expressed tissue-specifically in a chimericnon-human animal, an embryo of a strain deficient in a cell and/or atissue wherein the gene is expressed is employed as a host embryodescribed later. In the case of a gene that is constitutively expressed,an embryo of a strain deficient in any cell and/or tissue is employed asa host embryo described later.

A nucleic acid sequence (structural gene) encoding a desired proteinshould be located (ligated or inserted) in a way that the expression ofthe desired protein is regulated by the regulatory region of a gene thatis expressed in at least a specific cell and/or tissue. Hence, thenucleic acid sequence is located downstream of the regulatory region ofa gene that is expressed in a specific cell and/or tissue.

Alternatively, the nucleic acid sequence encoding the desired protein islocated downstream of the internal ribosomal entry site (IRES), that islocated between the termination codon and a sequence encoding a polyAsignal region of a gene that is expressed in a specific cell and/ortissue. Specifically, the nucleic acid sequence is in a state of beingfunctionally ligated to the IRES on the genome, and is present betweenthe termination codon and a sequence encoding a polyA signal region of agene that is expressed in a specific cell and/or tissue. Uponconstruction of a knock-in vector, the polyA signal region of a genethat is expressed in the above specific cell and/or tissue can be used,but other polyA signal regions can be used as well. For example, otherpolyA sequences known in the art such as a polyA signal region derivedfrom simian virus 40 (SV40) can also be used.

The nucleic acid sequence encoding a desired protein is located betweenthe termination codon and a sequence encoding a polyA signal region of agene that is expressed in a specific cell and/or tissue, and thesequence is inserted in the form that the sequence is located downstreamof a promoter sequence that is located downstream of a sequence encodinga 2nd polyA signal region. Specifically, the nucleic acid sequence ispresent in a state of being functionally ligated to the promotersequence and the sequence encoding the polyA signal region on thegenome, and the gene that is expressed in a specific cell and/or tissueoriginally existing on the genome is also present in a state of beingfunctionally ligated to the promoter sequence and the sequence encodingthe polyA signal region. Upon construction of a knock-in vector,examples of a promoter sequence used herein are not specificallylimited, as long as they are capable of regulating the expression in aspecific cell and/or tissue. Preferably, the promoter of a gene that isexpressed in a specific cell and/or tissue is used. When 2 promoters arepresent in a knock-in vector, the two promoters may be the same ordiffer from each other, as long as they are capable of regulating theexpression in the same cell and/or tissue. Furthermore, examples of asequence encoding a polyA signal region used for the construction of aknock-in vector are not specifically limited, as long as the polyAsignal region is known in the art, and include a polyA signal regionderived from the same origin as that of a promoter and a polyA signalregion derived from simian virus 40 (SV40). Furthermore, similarly tothe case of a promoter, when sequences encoding 2 polyA signal regionsare present in a knock-in vector, the two may be the same or differ fromeach other.

Moreover, the above nucleic acid sequence encoding a desired protein canalso be located in the order of a promoter sequence, the nucleic acidsequence, and a sequence encoding a polyA signal region downstream ofthe polyA signal region of a gene that is expressed in a specific celland/or tissue. Specifically the nucleic acid sequence is present in astate (cassette form) of being functionally ligated to a promoter and asequence encoding a polyA signal region, downstream of the polyA signalof a gene that is expressed in a specific cell and/or tissue. Withreference to construction of a knock-in vector, examples of a promotersequence used herein are not specifically limited, as long as they arecapable of regulating the expression in a specific cell and/or tissue.Preferably, the promoter of a gene that is expressed in a specific celland/or tissue is used. Furthermore, with reference to construction of aknock-in vector, examples of a sequence encoding a polyA signal regionare not specifically limited, as long as the polyA signal region isknown in the art, and include a polyA signal region derived from thesame origin as that of a promoter and a polyA signal region derived fromsimian virus 40 (SV40). In addition, when sequences encoding 2 polyAsignal regions are present in a knock-in vector, the two sequences maybe the same or differ from each other. The distance between the 3′ endof the polyA signal region of a gene that is expressed in a specificcell and/or tissue and the 5′ end of a promoter sequence that regulatesthe expression of a nucleic acid sequence encoding a desired protein isnot specifically limited, as long as the nucleic acid sequence can beexpressed in a specific cell and/or tissue. The longer distance thereofmay provide an unfavorable effect on the stability of mRNA, thetranscription product. In addition, this also leads to a largerstructure for a targeting vector, making it difficult to prepare avector with such a structure. Because of these reasons, the distancebetween the 3′ end of the polyA signal region and the 5′ end of thepromoter sequence regulating the expression of a nucleic acid sequenceencoding a desired protein is preferably within 1 Kb.

When a nucleic acid sequence (structural gene) encoding a desiredprotein is located downstream of the above regulatory region, thenucleic acid sequence may be inserted downstream of the regulatoryregion. Alternatively, for an allele on the one side of an ES cell, thenucleic acid sequence can also be simply located in a form whereby thesequence is located immediately following the regulatory sequence of agene that is expressed in a cell and/or tissue, so that the gene issubstituted with an original structural gene. The original structuralgene that has been substituted with the nucleic acid sequence encodingthe desired protein is expressed by an allele on the other side, so thatthe cell and/or tissue can maintain its normal conditions. However, in agene such as an immunoglobulin gene wherein allelic exclusion works, anIRES sequence is located following the termination codon of the originalstructural gene, and then the nucleic acid sequence encoding a desiredprotein can be located following the IRES sequence in alleles on bothsides of an ES cell. Moreover, an allele on one side of an originalstructural gene is previously inactivated in an ES cell, an IRESsequence is located following the termination codon of an originalstructural gene of an allele that has not been inactivated, and then thenucleic acid sequence encoding a desired protein can also be locatedfollowing the IRES sequence. In this case, the allele that has not beeninactivated is expressed exclusively, so that high-level expression ofthe nucleic acid sequence encoding a desired protein is expected at thesame time.

Expression of an immunoglobulin κ chain gene occurs by the binding ofmany V and J gene fragments by recombination as described above. As aresult of this binding, a promoter sequence existing in the vicinity ofthe upstream of each V gene fragment is located in the vicinity of anenhancer sequence that is present downstream of the J fragment. Anenhancer sequence can only activate the promoter in such a situation(Picard et al., Nature, 307: 80-2, 1984). Specifically, the abovenucleic acid sequence encoding a desired protein can also be located inthe vicinity of the enhancer sequence, while being artificially ligatedto the promoter sequence of an immunoglobulin κ chain gene. In theimmunoglobulin κ chain gene locus, another enhancer sequence is known tobe present downstream of the above enhancer sequence (Meyer et al., EMBOJ. 8: 1959-64, 1989). This gene is expressed at high levels in B cellsunder the influence of such multiple enhancers.

A pluripotent cell derived from a non-human animal comprising a genomewherein the above-mentioned nucleic acid sequence encoding a desiredprotein is located is hereinafter referred to as a knock-in cell or aknock-in ES cell. For example, these cells can be obtained as describedbelow, but a method for obtaining the cells is not limited thereto.

(1) Construction of a Targeting Vector (Knock-in Vector)

A knock-in vector wherein a nucleic acid sequence encoding a desiredprotein has been inserted downstream of a gene that is expressed in aspecific cell and/or tissue or a knock-in vector wherein a nucleic acidsequence encoding a desired protein is inserted downstream of aninternal ribosomal entry site that has been located downstream of a genethat is expressed in a specific cell and/or tissue are constructed.

A nucleic acid sequence to be introduced may be a cDNA or a genomic DNAcontaining introns, as long as it contains a region from the initiationcodon to the termination codon. A protein encoded by the nucleic acidsequence may be of any type. The nucleic acid sequence used in thepresent invention may be used for high-level expression or secretion ofthe protein encoded by the sequence, or may also be used for elucidatingthe functions of the protein. Therefore, any type of nucleic acidsequence to be introduced can be used, as long as the nucleotidesequence thereof is specified. Examples of a nucleic acid sequence(structural gene) include genes encoding functional proteins derivedfrom mammals, and preferably, humans, such as a gene encoding asecretory protein, a gene encoding a membrane protein, and a geneencoding intracellular or intranuclear protein.

An example of an IRES that can be utilized in the present invention is,but is not limited thereto, an IRES derived from encephalomyocarditisvirus (EMCV) (Jang et al., supra), pIREShyg (Clontech, U.S.A.). When anIRES and/or a nucleic acid sequence encoding a desired protein isinserted into a knock-in vector, an insertion site therefor ispreferably located between the termination codon and a polyA additionsite of a gene that is expressed in a specific cell and/or tissue. Inaddition, a plural number thereof may be inserted. To prevent thesecondary structure formed by an IRES sequence from having an adverseeffect on the translation of the above gene, it is preferable to providea certain space (e.g., several bp to several 10 bp) between thetermination codon gene of the above and the IRES.

To alter the genome of an animal so that it contains a nucleic acidsequence encoding a desired protein downstream of a gene that isexpressed in a specific cell and/or tissue, or so that is contains anIRES downstream of a gene that is expressed in a specific cell and/ortissue, and a nucleic acid sequence encoding the desired proteindownstream of the IRES, a knock-in vector is prepared. To this vectorDNA, the IRES and/or the nucleic acid sequence encoding a desiredprotein is inserted. Examples of a vector that can be used for thispurpose include plasmids and viruses. Persons skilled in the art caneasily select and obtain a vector that can be used as a knock-in vector.A specific example of a vector is, but is not limited to, pKIκ (seeexamples described later). In the knock-in vector, between thetermination codon and a polyA addition site (e.g., around the halfwaypoint) of a gene that is expressed in a specific cell and/or tissue, anappropriate restriction enzyme cleavage site is inserted as an insertionsite for an IRES sequence and/or a target nucleic acid sequence DNA. Inthe restriction enzyme cleavage site, a DNA (cDNA or genomic DNA)containing a region from the initiation codon to the termination codonof a nucleic acid sequence to be introduced is inserted. Furthermore,preferably, a translational stimulatory sequence such as a Kozaksequence can be located upstream of the initiation codon. Furthermore,the vector can contain a selection marker, for example, apuromycin-resistance gene, a neomycin-resistance gene, ablasticidin-resistance gene, or a GFP gene, if necessary.

(2) Introduction of Knock-in Vector into Pluripotent Cell Derived fromNon-Human Animal, and Selection of Homologous Recombinant

Pluripotent cells derived from a non-human animal can be transformedwith a knock-in vector according to a known technique in the art, forexample, the method described in Bio Manual Series 8, Gene Targeting,Shinichi Aizawa, YODOSHA, 1995. For example, the above-prepared knock-invector can be introduced into pluripotent cells by electroporation, thelipofection method, or the like.

In the step of preparing a knock-in (targeting) vector DNA, the ratio ofhomologous recombination to random insertion can be improved by treatingtargeting vector DNAs with polyamines or analogues thereof, or formingcomplexes of targeting vector DNAs and polyamines or analogues thereof.Such treatment with polyamines or analogues thereof, or the formation ofcomplexes with polyamines or analogues thereof, can be performed, beforetransformation, or before, during, or after the linearization of thetargeting vector, by bringing the vector into contact with at least onetype of polyamine or an analogue thereof. For example, an increase inthe ratio of homologous recombination is observed by adding 1 mMspermidine to a reaction buffer used when a knock-in vector islinearized.

The timing for bringing targeting vectors into contact with polyaminesor analogues thereof may be as described above in accordance with any ofthe following cases: polyamines or analogues thereof are added into asolution containing the vector before linearization of the vector;polyamines or analogues thereof are added into a restriction enzymebuffer for the linearization of the vector (e.g., 50 mM Tris-HCl, 10 mMMgCl₂, 100 mM NaCl, 1 mM Dithioerythritol); and/or polyamines oranalogues thereof are added to an HBS buffer (e.g., 25 mM Hepes, 137 mMNaCl, 5 mM KCl, 0.7 mM, Na₂HPO_(4.2)H₂O, 6 mM Dextrose) for dissolvingDNA after restriction enzyme treatment, phenol/chloroform treatment,ethanol precipitation, and dry treatment. In any of these cases, in anelectric pulse experiment, DNA to be added to cells is preferably in astate of forming a complex with a polyamine or an analogue thereof. Theconcentration for addition of polyamines or analogues thereof ispreferably between 0.1 mM and 10 mM, and is further preferably 1 mM.Addition at a concentration of I mM is used in a preferred embodiment ofthis invention. At a lower or a higher concentration than 1 mM, asimilar effect can be exhibited.

As representative polyamines other than spermidine (triamine), forexample, putrescine (diamine), cadaverine (diamine), and spermine(tetraamine) are known. These polyamines are known to have physiologicalaction similar to that of spermidine, specifically, 1) stabilization andaction causing conformational changes in a nucleic acid by interactionwith the nucleic acid, 2) promoting action of various nucleic acidsynthesis systems, 3) activation of protein synthesis, and 4)acetylation of histone, and the like. These polyamines are thought toexhibit effects analogous to that of spermidine. Known importantfeatures of polyamines are to form a complex with DNA, and to be apromoting factor in an in vitro recombination experimental system usingan enzyme derived from prokaryotic cells. Furthermore, it is also knownthat by the addition of polyamines to foreign DNA, the efficiency of theintroduction of the DNA into a mammalian cell is improved. In themeantime, polyamines have not conventionally been known to have aneffect on the ratio of homologous recombinants to clones with randominsertion that is an object of the present invention. The abovepolyamines are commercially available. For example, spermidine(manufactured by SIGMA, U.S.A.) is available. In addition, analogues ofpolyamines are not specifically limited, as long as they are compoundsthat can ionically bind to the phosphoric acids of DNA in a mannersimilar to that of polyamines. For example, poly-L-lysine andpoly-L-arginine are known. It is thought that these analogues alsoprovide stabilization and conformational changes in DNA by ionicallybinding to phosphoric acids of DNA, thereby exhibiting an effect similarto that provided by polyamines. Commercially available analogues can beused as the above analogues. For example, poly-L-lysine andpoly-L-arginine (manufactured by SIGMA, U.S.A.) are available.

As described above, by the utilization of polyamines or analoguesthereof, the ratio of homologous recombination to random insertion canbe improved. This use is thought to also have an effect on genedisruption (gene knock-out) via homologous recombination. That is, thepresent invention provides a method for gene targeting that comprisestreating a targeting vector (knock-in vector or knock-out vector) withpolyamines or analogues thereof, or a method for gene targeting thatcomprises forming complexes of targeting vectors and polymines oranalogues thereof.

The present invention further provides a reagent or a compositioncomprising at least 1 type of polyamine or analogue thereof to be usedfor the gene targeting method. Examples of such polyamines or analoguesthereof include, but are not limited to, spermidine, cadaverine,putrescine, spermine, poly-L-lysine, and poly-L-arginine. A preferredexample is spermidine. When pluripotent cells derived from a non-humananimal are transformed according to the present invention using atargeting vector treated with polyamines or analogues thereof asdescribed above, or a targeting vector that forms complexes withpolyamines or analogues thereof, the ratio of homologous recombinants toclones with random insertion can be improved compared with conventionalcases (see Example 11).

Furthermore, the structure of a targeting vector (knock-in vector orknock-out vector) can be altered to increase the efficiency ofhomologous recombination. Specifically, the efficiency of homologousrecombination can be increased by processing the targeting vector so asto prevent a negative selection marker used for eliminating a cellhaving a targeting vector randomly inserted in the genome from beingexposed at the end portion of the vector when the targeting vector islinearized.

Specifically, in the linearized targeting vector, it is preferred toprocess so that the 5′ end and the 3′ end of a gene structurefunctioning as a negative selection marker are located at least 1 Kb,and preferably 2 Kb or more, away from the 5′ end and 3′ ends of thetargeting vector, respectively. In general, since a region forhomologous recombination with a genome (homologous recombination region)is located on either the 5′ or the 3′ end of a negative selectionmarker, the distance from the vector end is 3 Kb or more in most cases.Furthermore, the other end of the negative selection marker is adjacentto the end of the vector in most cases. In the present invention, oneend of the negative selection marker, to which no homologousrecombination region is adjacent, is processed to provide a distance ofat least 1 kb from the terminus of a linearized vector, therebyincreasing the efficiency of homologous recombination. As a sequence tosecure the distance from the vector end, a plasmid sequence such as pUCused for the construction of a targeting vector can be kept intact (thatis, so that this sequence is not deleted upon linearization) and thenutilized (e.g., see FIGS. 1 to 7). Alternatively, any new non-codingsequence that is not homologous to a targeting region of interest can belocated adjacent to the negative selection marker. When linearization ofa vector is carried out, restriction enzyme recognition sites of atargeting vector used in this case are thoroughly examined to select anappropriate restriction enzyme site with which a distance between thevector end and the end of the negative selection marker can be secured.Then the vector is linearized, so that an effect of improving theefficiency of homologous recombination can be achieved. In addition,when such an appropriate restriction enzyme site cannot be found, anappropriate restriction enzyme recognition sequence can be introduced ata desired position in the targeting vector by a technique using PCR(Akiyama et al., Nucleic Acids Research, 2000, Vol. 28, No.16, E77.).

It is thought that the use of the above-described targeting vectorstructure results in decreased frequency of attacks on the negativeselection marker within cells by nuclease, and increases the efficiencyof homologous recombination.

Specifically, the present invention provides a gene targeting vectorwherein the 5′ end and the 3′ end of a gene structure functioning as anegative selection marker are located at least 1 Kb, and preferably 3 Kbor more, away from the 5′ end and the 3′ end, respectively, of thelinearized targeting vector, and a method for gene targeting, which usesthe targeting vector. In the above targeting vector, as a negativeselection marker, any known marker can be utilized. A preferred negativeselection marker is a diphtheria toxin A gene.

To conveniently identify homologous recombinants, a drug resistance genemarker can be previously inserted into a position, at which a foreigngene is knocked in. For example, TT2F mouse ES cells used in theexamples of this specification are derived from F1 individuals obtainedby the crossing of a C57BL/6 strain with a CBA strain. As describedabove (Deng & Capecchi, Mol. Cell. Biol., 12: 3365-71, 1992), when asequence of a genomic homologous region contained in a knock-in vectoris derived from C57BL/6, it is predicted that homologous recombinationwill occur at a higher rate in an allele derived from C57BL/6 in TT2Fcells. Specifically, from the start, a targeting vector (knock-invector) containing C57BL/6-derived genomic DNA is used, and, forexample, a G418-resistance marker can be inserted into a C57BL/6-derivedallele. Next, a knock-in vector containing a puromycin-resistance markerand the C57BL/6-derived genomic DNA is introduced into the obtainedG418-resistant strain, so that a puromycin-resistant and G418-sensitivestrain can be obtained. In this strain, the G418-resistance gene hasbeen removed by homologous recombination of the knock-in vector with thegene that is expressed in the above specific cell and/or tissue, andinstead, a structural gene encoding a desired protein and the puromycinresistance marker have been inserted. With such a method, the trouble ofSouthern analysis or the like in identification of homologousrecombinants can be avoided.

In a manner similar to the method described in PCT internationalapplication WO 00/10383 pamphlet filed by this applicant (internationalpublication, Mar. 2, 2000), puromycin-resistant clones are picked up,genomic DNA is prepared, and then homologous recombinants can beidentified by the Southern analysis method. The puromycin-resistancegene in the knock-in vector is derived from a Lox-P Puro plasmiddescribed in the WO 00/10383 pamphlet, and contains in forward directionLox-P sequences on both ends. Thus, by the method described in the WO00/10383 pamphlet, this resistance gene can be removed from pluripotentcells into which the gene has been knocked-in.

The above-mentioned knock-in vector, and techniques and means forimproving the efficiency of homologous recombination, can be applied forall the cells into which a gene can be introduced, and the use thereofis not limited to the production of chimeric animals. For example, ingene therapy directed to humans and human cells (e.g., blood cells andimmunocytes), the knock-in vector and the technique for improving theefficiency of homologous recombination described in this specificationcan be used for disrupting or introducing a desired gene.

2. Host Embryo Deficient in Specific Cell and/or Tissue

Next, in the method for producing chimeric non-human animals of thepresent invention, a host embryo (hereinafter also referred to as adeficient host embryo) of non-human animal strain which is deficient ina specific cell and/or tissue is prepared. Examples of such a deficienthost embryo include, when an immunoglobulin light chain gene is utilizedas a regulatory region, an embryo deficient in B-cells due to theknock-out of an immunoglobulin heavy chain gene (Tomizuka et al., Proc.Natl. Acad. Sci. U.S.A., 18: 722-727, 2000); when a T cell receptor geneis utilized as a regulatory region, an embryo deficient in T-lymphocytesdue to deficiency in the T cell receptor β chain (Mombaerts et al.,Nature, 360: 225-227, 1992); when a myoglobin gene is utilized as aregulatory region, an embryo deficient in muscle tissue due to theknock-out of a myogenin gene (Nabeshima et al., Nature, 364: 532-535,1993); when a crystallin gene is used as a regulatory region, an embryoderived from an aphakia (ak) strain that is a mutant of a mousedeficient in lens (Liegeois et al., Proc. Natl. Acad. Sci. U.S.A., 93:1303-1307, 1996), when a renin gene is utilized as a regulatory region,an embryo deficient in kidney tissue due to the knock-out of Sall1 gene(Nishinakamura et al., Development, 128: 3105-3115, 2001); when analbumin gene is utilized as a regulatory region, an embryo deficient inliver tissue due to c-Met gene deficiency (Bladt et al., Nature, 376:768-770, 1995); and when a lipase gene is used as a regulatory region,an embryo deficient in pancreas tissue due to the knock-out of a Pdx1gene (Jonsson et al., Nature, 371: 606-9, 1994). Preferred deficienthost embryos are as exemplified above, but deficient host embryos thatcan be used in the present invention are not limited to the aboveembryos.

Furthermore, for the selection of the time for development, geneticbackgrounds, and the like of host embryos in order to efficientlyproduce chimeric non-human animals, it is desired to use conditions thathave been previously examined for each ES cell line. For example, in thecase of a mouse, when a chimera is produced from TT2 cells or TT2F cells(wild-type color, Yagi et al., Analytical Biochemistry, 214: 70-76,1993) derived from CBA×C57BL/6 F1, the genetic background of a hostembryo is preferably Balb/c (white, CLEA JAPAN, INC., Japan), ICR(white, CELA JAPAN, INC., Japan) or MCH (ICR) (white, CLEA JAPAN, INC.,Japan). Hence, it is desired to use an embryo (e.g., an 8-cell-stageembryo) of a non-human animal obtained by back-crossing of the abovenon-human animal strains deficient in specific cells/and or tissues withthese strains as a deficient host embryo.

The deficient cell and/or tissue in a host embryo is complemented bypluripotent cells by blastocyst complementation (BC). Thus, the abovedeficient host embryo may be embryonic lethal, as long as it can developinto the blastocyst stage for the production of chimeric animals. Suchan embryonic-lethal embryo is produced principally at a probability ofone-fourth by crossing of animals having a gene deficiencyheterologously. Hence, by obtaining a plural number of embryos bycrossing, chimeric animals are produced according to the followingprocedures, and then animals wherein host embryos are deficient embryosare selected therefrom. This selection can be conducted by Southernanalysis, PCR analysis, or the like using DNA extracted from the bodytissues of the chimeric animals.

3. Production of Chimeric Embryo and Transplantation of the Embryo intoFoster Parent

Chimeric non-human animals can be produced from the knock-in (ES) celllines obtained in the above section “1. Preparation of pluripotentcells” by the method described by Shinichi Aizawa (supra) or the like.Specifically, the prepared knock-in pluripotent cells are injected intothe blastocysts or 8-cell-stage embryos of the deficient host embryosdescribed in the above section “2. Host embryo deficient in specificcell and/or tissue” using a capillary or the like. This embryonicblastocyst or the 8-cell-stage embryo is directly transplanted into theoviduct of a non-human animal that is a foster animal of the samespecies, or cultured for 1 day for the embryo to develop to ablastocyst, with the blastocyst then being transplanted into the uterusof the foster parent. Subsequently, the foster parents are fed to givebirth, thereby obtaining offsprings.

4. Expression of Transgene in Chimeric Non-Human Animal

The contribution ratio of the pluripotent cells in the offspringsderived from the knock-in pluripotent cell-injected embryos producedaccording to the above section “Production of chimeric embryo andtransplantation of the embryo into foster parent” can be roughlydetermined based on coat color. For example, when the knock-in cell linederived from TT2F cells (wild color) is injected into a host mouseembryo whose background is MCH(ICR) (white), the proportion of wildcolor (dark brown) to the others represents the contribution ratio ofthe pluripotent cells. Here the contribution ratio determined by coatcolor is correlated with the contribution ratio of knock-in pluripotentcells in cells and/or tissues other than the above deficient cell and/ortissue. However, in some tissues, the contribution ratio of knock-inpluripotent cells may not agree with the contribution ratio determinedby coat color. At the same time, in the above chimeric non-human animal,the above deficient cell and/or tissue derived from the host embryos isabsent, and only those derived from the knock-in pluripotent cells arepresent. Recovery of the deficient cell and/or tissue in the chimericnon-human animal by the contribution of the knock-in pluripotent cellscan be detected by FACS analysis (Fluorescence-Activated Cell Sorter),the ELISA method (Enzyme-Linked ImmunoSorbent Assay), or the like.Whether or not an inserted nucleic acid sequence (structural gene) isexpressed in a cell and/or tissue that is derived from the knock-inpluripotent cell is detected by the RT-PCR method using RNA derived fromthe cell and/or tissue (Kawasaki et al., P.N.A.S., 85: 5698-5702, 1988),the Northern blot method (Ausubel et al., Current protocols in molecularbiology, John Wiley & Sons, Inc., 1994), or the like. Expression at theprotein level can be detected utilizing the enzyme immunoassay (ELISA;Toyama/Ando, “Tan-kurohn Koutai Jikken Manual (Monoclonal AntibodyExperimental Manual),” Kodansha Scientific, 1987), the Western blotmethod (Ausubel et al., supra), or the like using chimeric mouse serum,when a specific antibody against a desired protein encoded by anintroduced nucleic acid sequence is already present. Moreover, by theprevious appropriate alteration of DNA encoding a nucleic acid sequence(structural gene) to be introduced, and addition of a tag peptide thatis detectable with an antibody to the DNA, the expression of theintroduced gene can be detected using the antibody or the like againstthe tag peptide (POD-labeled anti-His₆, Roche Diagnostics, Japan).

The chimeric non-human animal produced as described above expresses theintroduced nucleic acid sequence (structural gene) at high levels in atleast a specific cell and/or tissue. The thus expressed desired proteinis of a system secreting blood, milk, or the like, this system can beutilized as a production system of a useful protein. Furthermore, when aprotein with unknown functions is expressed at high levels, thefunctions of the protein can be elucidated based on the findingsobtained upon the above high level expression.

In recent years, the combination of the method for producing animalsfrom somatic-cell-nucleus-transferred embryos and gene targeting insomatic cells has further enabled gene alteration in animal species(e.g., cattle, sheep, and pigs) other than mice in a manner similar tothat in the case of mice (McCreath et al., Nature, 405: 1066-1069,2000). For example, cattle deficient in B cells can be produced byknock-out of immunoglobulin heavy chain. Moreover, a transgene isinserted downstream of the Ig gene locus of a mouse, cattle, sheep, pigor the like, an unfertilized egg is caused to develop wherein a nucleusof a fibroblast has been transferred, and then an ES cell can beprepared from an embryo at the blastocyst stage. A chimeric non-humananimal can be produced using the ES cell and the above B-cell-deficienthost embryo (Cibelli et al., Nature Biotechnol., 16: 642-646, 1998). Notonly in mice, but also in other animal species, high level expression ofa secretory protein is possible utilizing a similar expression system.By the use of large animals, this technique can be applied not only forthe analysis of gene functions, but also for the production of a usefulsubstance.

5. Production of Offspring of Chimeric Non-Human Animal

The method for producing chimeric non-human animals of the presentinvention further comprises obtaining by the selection of a transgenic(Tg) animal having heterologously a nucleic acid sequence that has beenintroduced by crossing the chimeric non-human animal with a non-humananimal of the same species, and obtaining an offspring of the Tg animalhaving the transgene homologously by crossing the male Tg animal withthe female Tg animal.

6. Tissue or Cell Derived from Chimeric Non-Human Animal or an OffspringThereof

In the present invention, tissues or cells derived from any of the abovechimeric non-human animal or the offspring thereof can be obtained. Thecell or tissue contains a genome wherein a nucleic acid sequenceencoding a desired protein is located so that the expression of thedesired protein is regulated by the regulatory region of a gene that isexpressed in the cell or tissue, and can express the desired protein.

Examples of the above tissue and cell include any tissue and cell, suchas B cells, the spleen, and the lymphoid tissue, as long as they arederived from a chimeric non-human animal or an offspring thereof, andcan express a desired protein.

The above tissue and cell can be collected and cultured by techniquesknown in the art. Whether or not the tissue and cell express a desiredprotein can also be confirmed according to a routine technique. Suchtissues and cells are useful in preparation of a hybridoma and inproduction of a protein, as described below.

7. Preparation of Hybridoma

In the present invention, a hybridoma can be obtained by fusing a cellof a chimeric non-human animal expressing a nucleic acid sequenceencoding an introduced desired protein in particular, a B cell or a cellobtained from the spleen containing B cells or from the lymphoid tissuewith a tumor cell capable of proliferation (e.g., myeloma cell). Amethod for preparing a hybridoma can be based on, for example, atechnique described in Ando, Chiba, “Tan-kurohn Koutai Jikken SousaNyuumon (Monoclonal Antibody Experimentation and ManipulationIntroduction),” Kodansha Scientific, 1991.

As a myeloma, a cell incapable of producing an autoantibody derived froma mammal, such as a mouse, a rat, a guinea pig, a hamster, a rabbit, ora human can be used. In general, an established cell line obtained frommice, for example, 8-azaguanine-resistant mouse (derived from BALB/c)myeloma cell lines P3X63Ag8U.1 (P3-U1) [Yelton, D. E. et al., CurrentTopics in Microbiology and Immunology, 81: 1-7 (1978)],P3/NSI/1-Ag4-1(NS-1) [Kohler, G. et al., European J. Immunology, 6:511-519 (1976)], Sp2/O-Ag14(SP-2) [Shulman, M. et al., Nature, 276:269-270 (1978)], P3X63Ag8.653(653) [Kearney, J. F. et al., J.Immunology, 123: 1548-1550 (1979)], P3X63Ag8(X63) [Horibata, K. andHarris, A. W., Nature, 256: 495-497 (1975)] and the like are preferablyused. These cell lines are subcultured in an appropriate medium, forexample, a 8-azaguanine medium [the medium is prepared by adding8-azaguanine to a RPMI-1640 medium supplemented with glutamine,2-mercaptoethanol, gentamycin, and fetal calf serum (hereinafterreferred to as “FCS”)], Iscove's Modified Dulbecco's Medium (hereinafterreferred to as “IMDM”), or Dulbecco's Modified Eagle Medium (hereinafterreferred to as “DMEM”). 3 to 4 days before cell fusion, the cell linesare subcultured in a normal medium (e.g., DMEM medium containing 10%FCS), and 2×10⁷ or more cells are secured on the day of fusion.

Cells that can be used for the expression of a desired protein encodedby a nucleic acid sequence introduced within the cell are plasma cells(that is, plasmacytes) and their precursor cells, the lymphocytes. Thesecells may be obtained from any site of an individual, and can begenerally obtained from the spleen, the lymph node, the bone marrow, thetonsil, peripheral blood, or an appropriate combination thereof.Splenocytes are most generally used.

Currently, the most generally conducted means for fusing a splenocyteexpressing a desired protein encoded by an introduced nucleic acidsequence with a myeloma is a method using polyethylene glycol, which hasrelatively low cytotoxicity and with which the procedure for cell fusionis simple. Specifically, this can be conducted as follows. Splenocytesand myelomas are washed well in a serum-free medium (e.g., DMEM) orphosphate-buffered saline (generally referred to as PBS), and then mixedto achieve a ratio of splenocytes to myeloma cells of approximately 5:1to 10:1 in general, followed by centrifugation. The supernatant isremoved, and then the precipitated cell groups are loosened well.Subsequently, 1 ml of a serum-free medium containing 50% (w/v)polyethylene glycol (with a molecular weight of 1000-4000) is addeddropwise while agitating the solution. After 10 ml of a serum-freemedium has been slowly added, centrifugation is performed. Thesupernatant is discarded again, the precipitated cells are suspended inan appropriate volume of a normal medium (generally referred to as HATmedium) containing a hypoxanthine-aminopterin-thymidine fluid and humaninterleukin-6. The suspension is dispensed in each well of a cultureplate, and then the cells are cultured in the presence of 5% carbondioxide gas at 37° C. for approximately 2 weeks. The media areappropriately supplemented with HAT media during culture.

When myeloma cells are of a 8-azaguanine-resistant line, that is, a cellline deficient in hypoxanthine-guanine-phosphoribosyltransferase(HGPRT), the myeloma cells that have not fused, and the myeloma cellsthat have fused to each other are unable to survive in HAT-containingmedia. On the other hand, fused cells of splenocytes or hybridomas ofsplenocytes and myeloma cells can survive, but the fused cells ofsplenocytes have a limited lifetime. Therefore, by continuing culture inHAT-containing media, only hybridomas of splenocytes and myeloma cellscan survive.

Hybridomas producing the desired protein encoded by the above introducednucleic acid sequence can be selected from the obtained hybridomas byELISA screening using an antibody specific to the desired proteinencoded by the above introduced nucleic acid sequence.

8. Method for Producing Protein

The present invention further provides a method for producing a desiredprotein, comprising causing the production of a desired protein usingany of the above chimeric non-human animals or the offspring thereof,the above tissues or cells, or the above hybridomas, and collecting theproteins. Specifically, chimeric non-human animals or offsprings thereofare fed under conditions that enable the expression of an introducednucleic acid sequence encoding a desired protein. Then the proteins, theexpression products, can be collected from the blood, ascites, or thelike of the animal. Alternatively, the tissue or the cell derived from achimeric non-human animal or an offspring thereof, the same that hasbeen immobilized (e.g., hybridomas immobilized by fusion with myelomacells), or the like is cultured under conditions that enable theexpression of an introduced nucleic acid sequence encoding a desiredprotein. Then the proteins, the expression products, can be collectedfrom the culture product, the culture supernatant, or the like. Theexpression products can be collected according to a known method such ascentrifugation, and then purified by one type of, or a combination ofmultiple known methods such as ammonium sulfate fractionation, partitionchromatography, gel filtration chromatography, adsorbent chromatography(e.g., ion exchange chromatography, hydrophobic interactionchromatography, and hydrophilic chromatography), fractionated thin-layerchromatography, and HPLC.

9. Method for Analyzing in vivo Functions

The present invention further provides a method for analyzing the invivo functions of a desired protein or a gene encoding the desiredprotein, comprising comparing the phenotype of the above chimericnon-human animal or an offspring thereof with that of a chimericnon-human animal or the like that has been produced from a correspondingwild-type ES cell containing no nucleic acid sequence encoding thedesired protein, and then determining differences among the phenotypes.

According to this method, any trait that appears in vivo correspondingto gene transfer is detected by a physicochemical method, whereby the invivo functions of the introduced nucleic acid sequence or the proteincan be identified. For example, blood of a chimeric non-human animalproduced from an ES cell containing a nucleic acid sequence encoding adesired protein, or an offspring thereof, and blood of a chimericnon-human animal produced from a corresponding wild-type ES cellcontaining no nucleic acid sequence encoding the above desired proteinare collected and then analyzed using a blood cell counter. By comparingthe measured concentrations in blood of leucocytes, erythrocytes,platelets, and the like between the above 2 types of chimeric non-humananimals, the action of the desired protein encoded by the introducednucleic acid sequence on the proliferation and differentiation ofblood-cell-system cells can be examined. In examples described later,DNA encoding thrombopoietin (TPO) was used as an introduced nucleic acidsequence. In this case, significant increases in platelets (trait) wereobserved in chimeric mice.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the structure of a knock-in basic vector pKIκ. Cκ: mouseIgκ gene, IRES: internal ribosomal entry site, Sal I: cloning site, CκpolyA: mouse Igκ polyA signal, Puro^(r): puromycin-resistance gene,DT-A: diphtheria toxin A chain gene, and PUC18: cloning vector.

FIG. 2 shows the structure of a plasmid pΔCκNeo. pSTneoB:neomycin-resistance gene, DT-A: diphtheria toxin A chain gene, andpUC18: cloning vector.

FIG. 3 shows the structure of a knock-in vector pKI-I-1. Promoter 1:mouse Igκ promoter region gene 1, MCS: multi-cloning site, Cκ: mouse Igκgene, SV40 polyA: SV40 PolyA signal, Cκ polyA: mouse Igκ polyA signal,Puro: puromycin-resistance gene, DT-A: diphtheria toxin A chain gene,and pUC18: cloning vector.

FIG. 4 shows the structure of a knock-in vector pKI-I-2. Promoter 2:mouse Igκ promoter region gene 2, MCS: multi-cloning site, Cκ: mouse Igκgene, SV40 polyA: SV40 PolyA signal, Cκ polyA: mouse Igκ polyA signal,Puro: puromycin-resistance gene, DT-A: diphtheria toxin A chain gene,and pUC18: cloning vector.

FIG. 5 shows the structure of a knock-in vector pKI-II-1. Promoter 1:mouse Igκ promoter region gene 1, MCS: multi-cloning site, Cκ: mouse Igκgene, Cκ polyA: mouse Igκ polyA signal, Puro: puromycin-resistance gene,DT-A: diphtheria toxin A chain gene, and pUC18: cloning vector.

FIG. 6 shows the structure of a knock-in vector pKI-II-2. Promoter 2:mouse Igκ promoter region gene 2, MCS: multi-cloning site, Cκ: mouse Igκgene, Cκ polyA: mouse Igκ polyA signal, Puro: puromycin-resistance gene,DT-A: diphtheria toxin A chain gene, and pUC18: cloning vector.

FIG. 7 shows the structure of a vector for knocking out mouse FGF23,pBlueLAB-LoxP-Neo-DT-A-3′KO-5′KO. 5′KO: FGF23 knock-out vector 5′homologous region, Neo^(r): neomycin-resistance gene, EX1: FGF23 exon 1partial region (+60 to +211), 3′KO: FGF 23 knock-out vector 3′homologous region, DT-A: diphtheria toxin A chain gene, and pBluescript:cloning vector.

BEST MODE OF CARRYING OUT THE INVENTION

A preferred embodiment of the present invention will be described withthe system utilizing an immunoglobulin light chain gene as an example.

Immunoglobulin (Ig) is a protein that is produced in the largestquantity among the proteins secreted in serum. For example in a human,immunoglobulin accounts for 10% to 20% of serum proteins, and theconcentration thereof reaches 10 to 100 mg/ml. Ig is produced by Bcells, and is mainly produced in large quantities by plasma cells thatare the terminal form of the differentiation of B cells. Various factorssuch as high transcription activity in the Ig gene locus, stability ofmRNA, and the functions of plasma cells specialized in secretion andproduction of proteins contribute to high level expression of Ig.Furthermore, in an adult, B cells are developed in the bone marrow, andshift to lymphoid tissues throughout the entire body such as the spleen,Peyer's patches of the small intestine, and the lymph node as theymature. Accordingly, the product of a transgene that is produced underthe regulatory region of an Ig gene of a B cell is released into bloodor lymph in a manner similar to that of the case of Ig, and isimmediately spread out throughout the entire body. The present inventionhas the advantage of causing the expression of a nucleic acid sequence(structural gene) encoding a desired protein utilizing the Ig expressionsystem that provides such high-level expression.

A position into which a transgene is inserted for efficient expressionthereof is the Ig light chain, and preferably the κ light chain. Forexample, 95% of mouse immunoglobulins contain the κ light chain, andthere is only one constant region gene thereof. On the other hand, thelight chain λ is contained in 5% of mouse immunoglobulins, and there are4 different types of genes, and any one of them may be used. Inaddition, in heavy chain, there is a total of 7 types of constantregions: μ, γ (four types), α, and ε. When it is taken intoconsideration that a transgene is generally inserted into 1 position,use of the κ chain is preferred.

As an expression form, it is preferred that a further introduced nucleicacid sequence be expressed under conditions whereby a functional Iglight chain is produced. In an Ig gene locus, it is known that thefunctional expression of Ig suppresses the expression of the otherallele by the mechanism of allelic exclusion. It is thought that mRNAwould be made unstable when recombination fails, so that the terminationcodon appears upstream from the original termination codon. Hence, tomaximize the expression of an introduced nucleic acid sequence, a statewherein a functional Ig light chain and the introduced nucleic acidsequence (structural gene) are present in a single mRNA is preferred.

The chimeric non-human animal or an offspring thereof of the presentinvention preferably contains on the genome a gene wherein an IRES islocated downstream of the termination codon of a nucleic acid sequenceencoding an immunoglobulin light chain, and a nucleic acid sequence(transgene) encoding a desired protein is located downstream of theIRES. The insertion site of an IRES+transgene is preferably between thetermination codon and a polyA addition site of an Ig light chain, andthe number of such sites may be multiple. To prevent the secondarystructure formed by the IRES sequence from having an adverse effect onthe translation of an Ig light chain gene, it is desirable to provide acertain space (e.g., several bp to several 10 bp) between thetermination codon of the Ig light chain gene and the IRES.

To alter the genome of an animal so that the genome contains an IRESdownstream of an immunoglobulin light chain gene, and it contains anucleic acid sequence encoding a desired protein downstream of the IRES,a knock-in vector is prepared. Into this vector DNA, the IRES and thenucleic acid sequence encoding a desired protein are inserted. As thisvector, PKIκ (see Example 1) is preferred. In the knock-in vector, anappropriate restriction enzyme cleavage site is introduced as aninsertion site for insertion of the IRES sequence and the nucleic acidsequence (to be introduced) between the termination codon and the polyAaddition site (e.g., in the vicinity of the halfway point) of the lightchain, and a DNA (cDNA or genomic DNA) containing a region from theinitiation codon to the termination codon of the nucleic acid sequenceto be introduced is inserted into the restriction enzyme cleavage site.In addition, preferably, a translational stimulatory sequence, such as aKozak sequence, can be located upstream of the initiation codon.Furthermore, for the simple identification of homologous recombinants, adrug resistance gene marker, and preferably a puromycin resistance gene,can be previously inserted into the position at which a foreign gene isknocked-in. In this step of preparing a knock-in vector DNA, treatmentwith polyamines such as spermidine or an analogue thereof is preferablyconducted.

Transformation of a non-human animal ES cell using a knock-in vector canbe performed by the method described by Shinichi Aizawa (supra) or thelike. In a manner similar to the method described in the PCTInternational Application WO 00/10383 pamphlet filed by the applicant(international publication on Mar. 2, 2000), puromycin-resistant clonesare picked up, genomic DNA is prepared, and then homologous recombinantsare identified by the Southern analysis method. The puromycin-resistancegene in the knock-in vector is derived from a Lox-P Puro plasmiddescribed in the WO 00/10383 pamphlet, and contains in the forwarddirection a Lox-P sequence at both of its ends. Hence, by the methoddescribed in the WO 00/10383 pamphlet, this resistance gene can beremoved from the ES cell to which the gene has been knocked-in.

When an immunoglobulin light chain gene is utilized in the presentinvention, as a deficient host embryo for injection of ES cells, anon-human animal strain which is homologous in disruption of theimmunoglobulin heavy chain gene described in the WO 00/10383 pamphlet ispreferably used.

The prepared knock-in ES cell is injected into the blastocyst or the8-cell-stage embryo of the above deficient host embryo using a capillaryor the like. This blastocyst or the 8-cell-stage embryo is directlytransplanted into the oviduct of a foster parent non-human animal of thesame species, or cultured for 1 day for the embryo to develop to ablastocyst, and then, the blastocyst is transplanted into the uterus ofa foster parent. Subsequently, the foster parents are fed to give birth,thereby obtaining offsprings.

In a chimeric non-human animal, host-embryo-derived mature B lymphocytesare absent, and only those derived from the knock-in ES cells arepresent. This is because the immunoglobulin heavy chain knock-outnon-human animal that is used as a host embryo is deficient in mature Blymphocytes (B220 positive), so that no immunoglobulins are detected inblood (Tomizuka et al., Proc. Natl. Acad. Sci. U.S.A., 97: 722-727,2000). Recovery of mature B lymphocytes and the production of antibodiesin the chimeric non-human animal due to the contribution of the knock-inES cells can be detected by the FACS analysis, ELISA method, or thelike. Whether or not a nucleic acid sequence inserted into the B cellderived from a knock-in ES cell is expressed depends on whether or not asite-specific recombination reaction takes place in an Ig light chaingene of the allele into which the sequence has been inserted.Specifically, when the Ig light chain gene of the inserted allele isrecombined successfully, and the mRNA thereof encodes a functional lightchain, the inserted nucleic acid sequence (structural gene) that ispresent at the same time on the mRNA is also translated into a proteinby the action of an IRES. Furthermore, when recombination of the κ chaingene of the inserted allele fails, and a κ chain or a λ chain on theother allele encodes functional light chain, transcription of mRNAencoding an unfunctional κ chain and the introduced nucleic acidsequence takes place, so that the protein derived from the insertednucleic acid sequence is expressed. The inserted nucleic acid sequenceis not expressed-when a κ chain or a λ chain of the other allelepreviously succeeds in functional recombination, and the recombinationof an Igκ chain of the inserted allele is shut off by the mechanism ofallelic exclusion. In a non-human animal, B cells appear in the fetalliver tissue around on day 12 of the prenatal period, and the place forthe development of B cells shifts to the bone marrow after birth. In thefetal period, B cells are in an early developmental period; that is,they mainly comprise cells expressing membrane immunoglobulin receptors.Because the number of B cells themselves is lower than in the case of anadult, and the quantity of mRNA encoding an immunoglobulin is low incells mainly expressing membrane Ig, it is thought that the expressionof the inserted nucleic acid sequence is also at a very low levelcompared with the case of an adult. Antibody production begins toincrease at the weanling period (3 weeks old), and this is inferred tobe due to increased plasma cells at the terminal differentiation stageof B cells. Subsequently, B cells shift to the lymphoid tissue such asthe spleen, the lymph node, and Payer's patches of the small intestine,and then express antibodies and the inserted nucleic acid sequence. Thedesired protein encoded by the inserted nucleic acid sequence issecreted in blood or lymph to spread throughout the entire body, in amanner similar to that of immunoglobulin.

The expression of an introduced nucleic acid sequence in B cells isconfirmed as described below. The expression of polycistronic mRNAcontaining the transgene is detected by the RT-PCR method, the Northernblot method, or the like using mRNA derived from a tissue or a cellpopulation, such as a spleen containing B cells and peripheral bloodnucleated cells. The expression at the protein level can be detectedutilizing enzyme immunoassay, the Western blot method, or the like usingchimeric mouse serum, when a specific antibody against a desired proteinencoded by the introduced nucleic acid sequence is already present. Inaddition, if the DNA encoding a introduced nucleic acid sequence isaltered properly, and a sequence encoding tag peptide detectable with anantibody is added to the DNA, the expression of the introduced nucleicacid sequence can be detected using an antibody or the like against thetag peptide.

The chimeric non-human animal obtained as described above expresses theintroduced nucleic acid sequence encoding the desired proteinefficiently and reliably at high levels. This high efficiency is asdescribed above, and is mainly based on the following points:

-   -   (1) By the use of B-lymphocyte-deficient host embryos, all the B        lymphocytes of a chimeric animal are derived from ES cells        regardless of chimerism.    -   (2) Homologous recombination occurs in an Igκ gene locus at an        efficiency of 5% or more.    -   (3) An expression system of immunoglobulins is utilized.    -   (4) The expression of immunoglobulin is very low in the early        developmental stage, and shows an explosive increase on and        after weanling stage. Thus, even when a gene whose high level        expression causes embryonic lethality is introduced, the        functions of the gene in an adult can be examined.

EXAMPLE

The present invention will be explained specifically in the followingexamples, but is not limited to these examples.

Example 1 Construction of Knock-in Vector pKIκ

(1) Preparation of a Fragment in the Vicinity of a Cloning Site

A restriction enzyme recognition sequence (Sal I recognition sequence)for the insertion of an internal ribosomal entry site (IRES) and anucleic acid sequence encoding a desired protein (to be introduced) isintroduced between the termination codon portion and a polyadenylationsignal (PolyA signal) of a mouse immunoglobulin kappa chain (Igκ) gene.Then a puromycin-resistance gene is inserted downstream of the PolyAsignal, thereby preparing a genomic fragment. The method is specificallydescribed below.

(1.1) Preparation of Upstream Fragment of a Cloning Site

The following DNAs were synthesized from the nucleotide sequenceencoding a mouse IgGκ gene obtained from GenBank (NCBI, U.S.A.). (SEQ IDNO: 1) igkc1: atctcgaggaaccactttcctgaggacacagtgatagg (SEQ ID NO: 2)igkc2: atgaattcctaacactcattcctgttgaagctcttgac

An Xho I recognition sequence was added to the terminus of igkc1 thatwas the 5′ primer, and an EcoR I recognition sequence was added to igkc2that was the 3′ primer. A reaction solution was prepared using TakaRaLA-Taq (TAKARA BIO INC., Japan) according to the instruction by themanufacturer. To 50 μL of the reaction solution, 10 pmol of each primer,and 25 ng of pBluescript SKII(+) (TOYOBO, Japan) to which aλ-clone-derived DNA fragment containing Ig light chain Cκ-Jκ had beencloned (WO 00/10383 pamphlet) as a template was added. The solution waskept at 94° C. for 1 minute, and then subjected to 25 cycles ofamplification, each cycle consisting of 94° C. for 30 seconds and 68° C.for 3 minutes. The obtained reaction solution was subjected tophenol/chloroform extraction, ethanol precipitation, and then digestionwith EcoR I and Xho I. DNA fragments were separated on 0.8% agarose gelelectrophoresis, target fragments were collected using GENECLEAN II Kit(Qbiogene, Inc., U.S.A.), and then an amplification fragment A wasobtained. After digestion with EcoR I and Xho I, the amplificationfragment A was inserted into a pBluescript2 KS-vector (Stratagene,U.S.A.) that had been dephosphorylated with Escherichia coli alkalinephosphatase, and then the vector was transformed into Escherichia coliDH5α. DNA was prepared from the obtained transformant, and then thenucleotide sequence was confirmed, thereby obtaining a plasmid pIgCκA.

(1.2) Preparation of Downstream Fragment of Cloning Site

The following DNAs were synthesized from the nucleotide sequenceencoding a mouse IgGκ gene obtained from GenBank (NCBI, U.S.A.). (SEQ IDNO: 3) igkc3: atgaattcagacaaaggtcctgagacgccacc (SEQ ID NO: 4) igkc4:atggatcctcgagtcgactggatttcagggcaactaaacatt

An EcoR I recognition sequence was added to the terminus of igkc3, whichwas the 5′ primer, and BamH I, Xho I, and Sal I recognition sequenceswere added to the 5′ side of igkc4, which was the 3′ primer. A reactionsolution was prepared using TakaRa LA-Taq (TAKARA BIO INC., Japan)according to the instructions by the manufacturer. To 50 μL of thereaction solution, 10 pmol of each primer, and 25 ng of pBluescriptSKII(+) (TOYOBO, Japan) to which a λ-clone-derived DNA fragmentcontaining Ig light chain Cκ-Jκ had been cloned (WO 00/10383 pamphlet)as a template was added. The solution was kept at 94° C. for 1 minute,and then subjected to 25 cycles of amplification, each cycle consistingof 94° C. for 30 seconds, 55° C. for 30 seconds, and 72° C. for 1minute. The obtained reaction solution was subjected tophenol/chloroform extraction, ethanol precipitation, and then digestionwith EcoR I and Bam HI. DNA fragments were separated on 0.8% agarose gelelectrophoresis, target fragments were collected using GENECLEAN II Kit(Qbiogene, Inc., U.S.A.), and then an amplification fragment B wasobtained. After digestion with EcoR I and BamH I, the amplificationfragment B was inserted into a pIgCκA vector that had beendephosphorylated with Escherichia coli alkaline phosphatase, and thenthe vector was transformed into Escherichia coli DH5α. DNA was preparedfrom the obtained transformant, and then the nucleotide sequence wasconfirmed, thereby obtaining a plasmid pIgCκAB.

(1.3) Insertion of Puromycin-Resistance Gene

Lox-P Puro plasmid (WO 00/10383 pamphlet) was digested with EcoR I andXho I, and then converted to blunt ends with T4 DNA polymerase. DNAfragments were separated on 0.8% agarose gel electrophoresis, and thenDNA fragments containing loxP-puromycin-resistance genes were collectedusing GENECLEAN II Kit (Qbiogene, Inc., U.S.A.). The obtainedloxP-puromycin-resistance gene fragment was inserted into the pIgCκABplasmid that had been digested with Sal I and converted into blunt ends,and then the plasmid was transformed into Escherichia coli DH5α. DNA wasprepared from the obtained transformant, and then the nucleotidesequence of the ligated portion was confirmed, thereby obtaining aplasmid pIgCκABP.

(1.4) Insertion of IRES Gene

The following DNAs were synthesized from the nucleotide sequenceencoding an IRES gene derived from encephalomyocarditis virus obtainedfrom GenBank (NCBI, U.S.A.). (SEQ ID NO: 5) eIRESFW:atgaattcgcccctctccctccccccccccta (SEQ ID NO: 6) esIRESRV:atgaattcgtcgacttgtggcaagcttatcatcgtgtt

An EcoR I recognition sequence was added to the terminus of eIRESFW,which was the 5′ primer, and EcoR I and Sal I recognition sequences wereadded to the 5′ side of esIRESRV, which was the 3′ primer. A reactionsolution was prepared using TakaRa LA-Taq (TAKARA BIO INC., Ltd, Japan)according to the instructions by manufacturer. To 50 μL of the reactionsolution, 10 pmol of each primer and 150 ng of the pIREShyg plasmid(Clontech, U.S.A.) as a template were added. The solution was kept at94° C. for 1 minute, and then subjected to 25 cycles of amplification,each cycle consisting of 94° C. for 30 seconds, 55° C. for 30 seconds,and 72° C. for 1 minute. The obtained reaction solution was subjected to0.8% agarose gel electrophoresis so as to separate DNA fragments, andtarget fragments were collected using GENECLEAN II Kit (Qbiogene, Inc.,U.S.A.). The obtained DNA fragment was inserted into the pGEM-T vector(Promega, U.S.A.), and then the vector was transformed into Escherichiacoli DH5α. DNA was prepared from the obtained transformant, and then thenucleotide sequence was confirmed, thereby obtaining a plasmidIRES-Sal/pGEM. This plasmid was digested with EcoR I, DNA fragments wereseparated on 0.8% agarose gel electrophoresis, and then target fragmentswere collected using GENECLEAN II Kit (Qbiogene, Inc., U.S.A.). Theobtained IRES gene was inserted into the pIgCκABP plasmid digested withEcoR I, and then the plasmid was transformed into Escherichia coli DH5α.DNA was prepared from the obtained transformant, and then the nucleotidesequence of the ligation portion was confirmed, thereby obtaining aplasmid pIgCκABPIRES.

(2) Preparation of pΔCκSal Plasmid

After digestion of a targeting vector plasmid for immunoglobulin genelight chain κ knock-out described in the WO 00/10383 pamphlet with SacII, partial digestion treatment with EcoR I was performed. The remaining14.6 Kb of DNA after excision of the LoxP-PGKPuro portion was separatedon 0.8% agarose gel electrophoresis, and then collected using GENECLEANII Kit (Qbiogene, Inc., U.S.A.). By insertion of the following syntheticDNAs into the obtained DNA, a Sal I recognition sequence was introduced.Sal 1 plus: agtcgaca (SEQ ID NO: 7) Sal 1 minus: aatttgtcgactgc (SEQ IDNO: 8)

The obtained plasmid was transformed into Escherichia coli DH5α, andthen DNA was prepared from the obtained transformant, thereby obtaininga plasmid pΔCκSal.

(3) Construction of Knock-in Vector pKIκ

The pIgCκABPIRES plasmid obtained in (1.4) above was digested with XhoI, DNA fragments were separated on 0.8% agarose gel electrophoresis, andthen DNA fragments containing Cκ-IRES-loxP-puromycin-resistance geneswere collected using GENECLEAN II Kit (Qbiogene, Inc., U.S.A.). ThepCκSal prepared in (2) above was digested with Sal I, and thendephosphorylated with Escherichia coli alkaline phosphatase. Into theresultant pCκSal, the above DNA fragment was inserted, and then theplasmid was transformed into Escherichia coli DH5α. DNA was preparedfrom the obtained transformant, and then the nucleotide sequence of theligated portion was confirmed, thereby obtaining a plasmid pKIκ. Thestructure of pKIκ is shown in FIG. 1. The outline of the pKIκ vectorstructure is as follows.

Specifically, the vector comprises, from the 5′ side, mouse Igκ genomicDNA containing the enhancer sequence existing downstream of a J fragmentand the Igκ light chain C region (EcoR I to EcoR I, approximately 5.3Kb), an internal ribosomal entry site (approximately 0.6 Kb), thecloning site (Sal I) located immediately following the 3′ end of theinternal ribosomal entry site, a mouse Cκ polyA signal region(approximately 0.3 Kb), a puromycin-resistance gene cassette(approximately 1.8 Kb), a mouse Igκ genomic DNA located downstream of amouse Cκ polyA signal region (approximately 7.7 Kb), a diphtheria toxinA (DT-A) gene cassette (approximately 1.0 Kb), and PUC18 DNA(approximately 2.7 Kb). Knock-in ES cells are prepared by conducting, ina similar manner, the procedures described in Example 2 and thefollowing examples described later by using the above described knock-invector. In the knock-in ES cell, an internal ribosomal entry site islocated immediately following the termination codon of the Cκ structuralgene of mouse Igκ, and a desired structural gene can be located at acloning site located immediately following the 3′ end of the internalribosomal entry site. Furthermore, a mouse Cκ polyA signal region can belocated immediately following the desired structural gene. By the use ofan IRES, the translational efficiency of a structural gene locateddownstream of the termination codon of the Cκ structural gene of mouseIgκ is improved drastically (Mizuguchi et al., Molecular Therapy Vol. 1,No. 4, 2000, 376-382.).

Example 2 Insertion of TPO Gene into pKIκ

(1) Preparation of Mouse Thrombopoietin (TPO) Gene for Insertion intopKIκ

The following DNAs were synthesized from the nucleotide sequenceencoding a mouse TPO gene (International Publication WO 95/18858pamphlet). (SEQ ID NO: 9) tpoN: CCGCTCGAGCGGCCACCATGGAGCTGACTGATTTGCT(SEQ ID NO: 10) tpoR: CCGCTCGAGCGGCTATGTTTCCTGAGACAAATTCC

An Xho I recognition sequence and a Kozak sequence were added to the 5′side of the terminus of tpoN, which was the 5′ primer, and Xho Irecognition sequence was added to the terminus of tpoR, which was the 3′primer.

A reaction solution was prepared using TaKaRa LA-Taq (TAKARA BIO INC.,Japan) according to the instructions by manufacturer. To 100 μL of thereaction solution, 10 pmol of each primer and 100 pg of Marathon-ReadycDNA (Mouse Liver, Clontech, U.S.A.) as a template were added. Thesolution was kept at 94° C. for 30 seconds, and then subjected to 25cycles of amplification, each cycle consisting of 98° C. for 1 second,50° C. for 30 seconds, and 72° C. for 1 minute. The obtained reactionsolution was subjected to 0.8% agarose gel electrophoresis, so as toseparate PCR amplification fragments, and then target DNA fragments werecollected using a QIAquick Gel Extraction Kit (QIAGEN, Germany). Theobtained DNA fragments were treated with Xho I at 37° C. for 3 hours,and then collected using the QIAquick PCR Purification Kit (QIAGEN,Germany). pBluescript II SK(−) (TOYOBO, Japan) was treated with Xho Iand then with CIAP (TAKARA BIO INC., Japan), thereby obtaining apBlueXhoICIAP plasmid. 42 ng of the pBlueXhoICIAP plasmid, 108 ng of thePCR amplification fragment obtained by treatment with Xho I, and 10 μLof a DNA Ligation Kit Ver. 2, Solution I (TAKARA BIO INC., Japan) weremixed. After incubation at 16° C. for 30 minutes, the product wastransformed into Escherichia coli DH5α. A plasmid DNA (pBlueTPO) wasprepared from the obtained transformant, and then the nucleotidesequence was confirmed. 9.6 μg of pBlueTPO was digested with Xho I at37° C. for 4 hours, and then TPO fragments were collected by 0.8%agarose gel electrophoresis using a QIAquick Gel Extraction Kit (QIAGEN,Germany). The collected TPO fragments were separated on 0.8% agarose geland collected again, thereby preparing a mouse TPO gene for insertioninto pKIκ.

(2) Preparation of Mouse TPO Gene-Inserted Knock-in Vector

21 μg of pKIκ prepared in Example 1 was digested with Sal I at 37° C.for 4 hours, and then vectors were collected by ethanol precipitation.The collected vector was dissolved in 10 μL of TE, and thenpKIκ-SalI-CIAP dephosphorylated with CIAP (TAKARA BIO INC., Japan) wasprepared. 40 ng of dephosphorylated pKIκ-SalI-CIAP, 4 ng of the mouseTPO gene for insertion into pKIκ prepared in (1) above, and 1.8 μL of aDNA Ligation Kit Ver. 2, Solution I (TAKARA BIO INC., Japan) were mixed,incubated at 16° C. for 18 hours, and then transformed into Escherichiacoli XL10-Gold Ultracompetent Cells (TOYOBO, Japan). DNA was preparedfrom the obtained transformant, the nucleotide sequence was confirmed,and then a mouse TPO gene-inserted knock-in vector was prepared using aQIAfilter Plasmid Kit (QIAGEN, Germany).

(3) Preparation of Mouse TPO Gene-Inserted Knock-in Vector forElectroporation

150 μg of the mouse TPO gene-inserted knock-in vector prepared in (2)above was digested with Xho I at 37° C. for 6.5 hours, and thensubjected to phenol/chloroform extraction. 2.5 volumes of 100% ethanoland 0.1 volume of 3M sodium acetate were added to the digest, and thenthe mixture was stored at −20° C. for 16 hours. The vectors werelinearized with Xho I, collected by centrifugation, and then sterilizedby the addition of 70% ethanol. 70% ethanol was removed in a cleanbench, and then air-dried for 1 hour. An HBS solution was added toobtain a 0.5 μg/μL DNA solution and then stored at room temperature for1 hour, so that a mouse TPO gene-inserted knock-in vector forelectroporation was prepared.

Example 3 Preparation of TPO Knock-in ES Cell Line

To prepare a mouse ES cell line wherein TPO-cDNA was inserted downstreamof an immunoglobulin κ light chain gene by homologous recombination, theTPO knock-in vector prepared in Example 2 was linearized with an Xho Irestriction enzyme (TAKARA BIO INC., Japan), and then transfected into aTT2F mouse ES cell (Yagi et al., Analytical Biochem., 214: 70, 1993) byan established method (Shinichi Aizawa, Bio Manual Series 8, GeneTargeting, YODOSHA, 1995).

The method for culturing TT2F was performed as described (ShinichiAizawa, supra). Feeder cells used herein were G418-resistant primarycultured cells (purchased from Invitrogen, Japan) treated with mitomycinC (SIGMA, U.S.A.). First, the propagated TT2F cells were trypsinized,and then suspended in HBS to achieve a concentration of 3×10⁷ cells/ml.0.5 ml of the cell suspension was admixed with 10 μg of the vector DNA,and then the mixture was subjected to electroporation using a GenePulser Cuvette (electrode distance: 0.4 cm; Bio-Rad, U.S.A.) (capacity:960 μF; voltage: 240 V, room temperature). The cells that had beensubjected to electroporation were suspended in 10 ml of an ES medium(Shinichi Aizawa, supra), and then inoculated onto a single 100 mmplastic dish for tissue culture (Falcon, Becton Dickinson, U.S.A.) intowhich feeder cells had been previously inoculated. 36 hours later, themedium was changed with an ES medium containing 0.8 μg/ml puromycin(SIGMA, U.S.A.). A total of 124 colonies were picked up from amongcolonies that had appeared 7 days later, and each colony was allowed togrow to reach confluence in a 24-well plate. Two-thirds of the colonieswere suspended in 0.2 ml of a medium for storage (ES culture media+10%DMSO, SIGMA, U.S.A.), and then stored at −80° C. The remaining one-thirdof the colonies were inoculated onto a 12-well gelatine-coated plate,and then cultured for 2 days, thereby preparing genomic DNA from 10⁶-10⁷cells using Puregene DNA Isolation Kits (Gentra Systems, U.S.A.). TheseG puromycin-resistant TT2F cell genomic DNAs were digested with arestriction enzyme EcoR I (TAKARA BIO INC., Japan), and then separatedby agarose gel electrophoresis. Subsequently, Southern blot wasperformed, and then homologous recombinants were detected using as aprobe a DNA fragment (Xho I to EcoR I; approximately 1.4 kb) of the 3′end of the Ig light chain Jκ-Cκ genomic DNA used in the proceduresdescribed in the WO 00/10383 pamphlet (see Example 48). As a result, 2out of 124 clones (2%) were homologous recombinants. In wild-type TT2Fcells, a single band was detected by digestion with EcoR I. It ispredicted that in the homologous recombinants, a new band will appearbelow the band (WO 00/10383 pamphlet (see Example 58)) in addition tothe single band. In the puromycin-resistant clones #6 and #11, this newband was confirmed. Hence, in these clones, TPO-cDNA had been inserteddownstream of immunoglobulin κ chain gene on one allele.

Example 4 Production of Chimeric Mouse using TPO Knock-in Mouse ES CellClone and Host Embryo Derived from B-lymphocyte-Deficient Mouse Strain

The homozygote of immunoglobulin μ chain gene knock-out is deficient infunctional B-lymphocytes, and no antibodies are produced (Kitamura etal., Nature, 350: 423-426, 1991). Embryos obtained by the crossing offemale and male mice of the above homozygotes bred in a cleanenvironment were utilized as hosts for the production of chimeric micein this example. In this case, most functional B-lymphocytes in chimericmice are derived from ES cells that have been injected. In this example,mice obtained by 3 or more backcrossings of the immunoglobulin μ chaingene knock-out mouse described in the report of Tomizuka et al., (Proc.Natl. Acad. Sci. U.S.A., 97: 722-7, 2000) and the mice of MCH (ICR)strain (CLEA JAPAN, INC., Japan) were used for preparing host embryos.

The puromycin-resistant TT2F cell clone #11 that had been obtained inthe above Example 3 and confirmed to have TPO-cDNA inserted downstreamof an immunoglobulin κ chain gene was prepared from frozen stock. 8 to10 of these cells were injected into a 8-cell-stage embryo obtained bythe crossing of the female and male mice of the above immunoglobulin μchain knock-out mouse homozygotes. The embryos were cultured overnightin ES media (Shinichi Aizawa, Bio Manual Series 8, Gene Targeting,YODOSHA, 1995) to develop into blastocysts. Approximately 10 injectionembryos were transplanted per uterus on the one side of a foster parentMCH (ICR) mouse (CLEA JAPAN, INC., Japan) at 2.5 days afterpseudopregnancy treatment. As a result of the transplantation of a totalof 180 injection embryos, 16 chimeric mouse offspring were born. Micewere determined to be chimeric if they had coat color wherein wild color(dark brown) derived from TT2F cells was observed with a white colorderived from the host embryos. 10 out of 16 offspring born had clearwild color portions in their coat colors, that is, wherein thecontribution of ES cells was observed. The highest contribution rate was100% observed in 2 mice. This result showed that the puromycin-resistantTT2F cell clone #11 having TPO-cDNA inserted downstream of theimmunoglobulin κ chain gene possessed chimera formation ability, thatis, had the capability of differentiating into a normal tissue of amouse.

Example 5 Increase of Platelet Counts in TPO Knock-in ES Cell-DerivedChimeric Mouse

Blood was collected from the retro-orbital sinus of a total of 10chimeric 6-week-old mice derived from TPO knock-in ES cell clone # 11prepared as in the above Example 4 (chimerism of 100% to 5%) and 5non-chimeric 6-week-old mice. The number of blood cells of peripheralblood was counted using a blood cell counter (manufactured by SYSMEXCORPORATION, Japan, F-820). In the chimeric mouse group, an average ofan 11.04-fold increase in the number of platelet counts was observedcompared with the case of non-chimeric mice, regardless of chimerism. Inthe chimeric mouse group, an average of a 2.03-fold increase in thenumber of leuckocytes was similarly observed; however, conversely, anaverage of a 0.76-fold decrease was observed in the number oferythrocyte counts.

Therefore, the protein encoded by TPO gene was thought to have afunction to control the number of platelet counts in blood. This resultis consistent with the conventionally shown function of TPO geneproducts (Kato et al., Department of Blood and Tumor (Ketsueki Shuyo-ka)33: 1-10, 1996). Hence, it was shown that the method described in thepresent invention is useful for analyzing the in vivo functions of agene and of gene products.

Example 6 Preparation of TPO-Producing Hybridoma from TPO Knock-in ESCell-Derived Chimeric Mouse

On week 10 after birth, the spleens were collected from the chimericmice TPO (113) (chimerism of 70%), for which increases in platelets hadbeen observed in the above Example 5. Cell fusion of splenocytes of thespleens and myeloma cells was conducted, thereby preparing hybridomas.The method for preparing hybridomas was conducted according to theestablished method (Ando, Introduction for Monoclonal AntibodyExperimental Protocols (Monoclonal Ko-tai Jikken So-sa Nyumon), KodanshaScientific, 1991), and SP2/0 (RIKEN GENE BANK, RCB0209) was used formyeloma cells. The thus prepared hybridomas were plated onto three96-well plates. After 10 days of culture, the culture supernatant wasanalyzed by the ELISA method. The ELISA method was conducted asdescribed in Nishiyama et al. (Thromb Haemost 85: 152-159, 2001). First,anti-mouse TPO rabbit IgG antibodies were incubated overnight at 4° C.to immobilize onto a microtiter plate (Sumilon, Sumitomo Bakelite,Japan), and then the plates were washed. Next, the hybridoma supernatantwas added to the plates, and the plates were incubated overnight at 4°C., and then washed. The mouse TPO was finally detected using abiotinylated anti-mouse TPO avian IgG antibody, an alkalinephosphatase-labeled streptavidin, and LumigenPPD (Wako, Japan). As aresult, expression of mouse TPO was detected in approximately 15% of thewells wherein HAT-resistant hybridomas were present. Furthermore, whenthe expression of Igκ chain was examined by the ELISA method,approximately 25% of the HAT-resistant wells was determined as Igκ chainpositive, and approximately 60% of IgK-positive wells was determined asmouse TPO positive. The TPO concentration in the cloned TPO-positivehybridoma supernatant was approximately 10 ng/ml when quantified by theabove-described ELISA method.

Example 7 Genetic Transmission of TPO Knock-in Immunoglobulin κ ChainGene Locus from TPO Knock-in ES Cell-Derived Chimeric Mouse

To determine whether offpsrings derived from ES cells were born by thecrossing of male MCH (ICR) strain mouse (CLEA Japan, Inc., Japan) with afemale mouse TPO (108) among the TPO knock-in ES cell clone #11-derivedchimeric mice produced in the above Example 4 which had shown 100%chimerism, an examination was carried out. By the crossing, wild coloroffspring mice were born from eggs derived from TT2F cells (wild color,dominance) in the chimeric mice and fertilized with sperms derived fromMCH (ICR) male mice (albino, recessive); white offspring mice were bornfrom eggs derived from MCH (ICR) in the chimeric mice and fertilizedwith sperms derived from MCH (ICR) male mice (albino, recessive). Allthe 7 offspring mice in total obtained by the crossings showed wildcolor that was coat color derived from ES cells, indicating theefficient transmission of TPO knock-in ES cells to the germ lines. Whenthe number of platelets was counted in a manner similar to that in theabove Example 5 for seven 3-week-old wild colored offspring mice,significant increases (5×10⁶/μl or more) in the number of platelets wereobserved in 3 out of 7 mice (43%). TPO-cDNA had been inserted into animmunoglobulin gene locus on only one allele in the ES cells. Thus, theprobability of carrying the TPO gene inserted in the wild colored miceis inferred to be 50%. The above result is roughly consistent with thisinference, showing the genetic transmission of the introduced TPO gene.The above result shows that according to the method described in thisspecification, a mouse strain expressing a desired secretory protein ina cell or a tissue that is different from the site at which the proteinis originally expressed can be established.

Example 8 Insertion of Human CD20 Gene to pKIκ

(1) Preparation of Human CD20 Gene for Insertion into pKIκ

The following DNAs were synthesized from the nucleotide sequenceencoding a human CD20 gene (Genbank, M27394). (SEQ ID NO: 11) 20+:CCCTCGAGCCACCATGACAACACCCAGAAATTCAG (SEQ ID NO: 12) 20−:GGGTCGACTTAAGGAGAGCTGTCATTTTC

The amplification of the human CD20 gene was carried out using QuickClone cDNA, human spleen (Clontech, U.S.A.) as a template in a mannersimilar to that in the above Example 2. The PCR reaction solution wassubjected to 0.8% agarose gel electrophoresis to separate PCRamplification fragments, and then target DNA fragments were collectedusing a QIAquick Gel Extraction Kit (QIAGEN, Germany). The obtained DNAfragments were treated with Xho I at 37° C. for 3 hours, and thencollected using a QIAquick PCR Purification Kit (QIAGEN, Germany).

pBluescript II SK(−) (TOYOBO, Japan) was treated with Xho I, and thenwith CIAP (TAKARA BIO INC., Japan). 42 ng of the thus obtainedpBlueXhoICIAP plasmid, 108 ng of the PCR amplification fragment obtainedby treatment with Xho I as described above, and 10 μL of a DNA LigationKit Ver. 2, Solution I (TAKARA BIO INC., Japan) were mixed, incubated at16° C. for 30 minutes, and then transformed into Escherichia coli DH5α.A plasmid DNA (pBlueCD20) was prepared from the obtained transformant,and then the nucleotide sequence was confirmed. 9.6 μg of pBlueCD20 wasdigested with Xho I at 37° C. for 4 hours, and then CD20 fragments werecollected by 0.8% agarose gel electrophoresis using a QIAquick GelExtraction Kit (QIAGEN, Germany). The collected CD20 fragments wereseparated on 0.8% agarose gel and collected again, thereby preparing ahuman CD20 gene for insertion into pKIκ.

(2) Preparation of Human CD20 Gene-Inserted Knock-in Vector

21 μg of pKIκ prepared in Example 1 was digested with Sal I at 37° C.for 4 hours, and then the vectors were collected by ethanolprecipitation. The collected vector was dissolved in 10 μL of TE, andthen pKIκ-SalI-CIAP was prepared by dephosphorylation with CIAP (TAKARABIO INC., Japan).

40 ng of the above KI-SalI-CIAP, 4 ng of the human CD20 gene forinsertion into a knock-in vector prepared in the above (1), and 1.8 μLof a DNA Ligation Kit Ver. 2, Solution I (TAKARA BIO INC., Japan) weremixed, incubated at 16° C. for 18 hours, and then transformed intoEscherichia coli XL10-Gold Ultracompetent Cells (TOYOBO, Japan). DNA wasprepared from the obtained transformant, and then the nucleotidesequence was confirmed. A human CD20 gene-inserted knock-in vector wasthen prepared using a QIAfilter Plasmid Kit (QIAGEN, Germany).

(3) Preparation of Human CD20 Gene-Inserted Knock-in Vector forElectroporation

150 μg of the human CD20 gene-inserted knock-in vector prepared in (2)was digested with Xho I (37° C., 6.5 hours) using a spermidine-added (1mM, pH 7.0, SIGMA, U.S.A.) buffer (Roche Diagnostics, Japan, H bufferfor restriction enzyme), or a spermidine-free buffer (Roche Diagnostics,Japan, H buffer for restriction enzyme). After phenol/chloroformextraction, 2.5 volumes of 100% ethanol and 0.1 volume of 3M sodiumacetate were added, and then the solution was stored at −20° C. for 16hours. The vector linearized with Xho I was collected by centrifugation,and then sterilized by the addition of 70% ethanol. 70% ethanol wasremoved in a clean bench, and then air-dried for 1 hour. HBS solutionwas added to result in a 0.5 μg/μL DNA solution, and the resultant wasstored at room temperature for 1 hour, thereby preparing a human CD20gene-inserted knock-in vector for electroporation.

Example 9 Preparation of Human CD20 Knock-in ES Cell Clone

To obtain a mouse ES cell clone wherein human CD20-cDNA has beeninserted by homologous recombination downstream of an immunoglobulin κlight chain gene, the 2 types of the human CD20 knock-in vectors(spermidine-added and spermidine-free) prepared in the above Example 8were transfected into each mouse ES cell TT2F (Yagi et al., AnalyticalBiochem., 214: 70, 1993) according to the established method (ShinichiAizawa, Bio Manual Series 8, Gene Targeting, YODOSHA, 1995). The methodfor culturing TT2F cells was performed according to the above-describedmethod (Shinichi Aizawa, supra). Feeder cells used herein wereG418-resistant primary cultured cells (purchased from Invitrogen, Japan)treated with mitomycin C (SIGMA, U.S.A.). First, the propagated TT2Fcells were trypsinized, and then suspended in HBS to be 3×10⁷ cells/ml.0.5 ml of the cell suspension was mixed with 10 μg of the vector DNA,and then the mixture was subjected to electroporation using a GenePulser Cuvette (electrode distance: 0.4 cm, Bio-Rad, U.S.A.) (capacity:960 μF; voltage: 240V; room temperature). The cells subjected toelectroporation were suspended in 10 ml of an ES medium, and theninoculated onto a 100 mm plastic dish for tissue culture (Falcon, BectonDickinson, U.S.A.), onto which feeder cells had been previouslyinoculated. 36 hours later, the medium was changed with an ES mediumcontaining 0.8 μg/ml puromycin (SIGMA, U.S.A.). Among the colonies thathad appeared 7 days later, 80 (spermidine-free) and 56(spermidine-added) colonies were picked up in total. Each type of thesecolonies was grown to reach confluence in a 24-well plate. Two-thirds ofthese colonies were suspended in 0.2 ml of a medium for storage (ESmedium+10% DMSO, SIGMA, U.S.A.), and then stored at −80° C. Theremaining one-third of the colonies were inoculated onto a 12-wellgelatine-coated plate, and then cultured for 2 days. Genomic DNA wasprepared from 10⁶ to 10⁷ cells using Puregene DNA Isolation Kits (GentraSystems, U.S.A.). These genomic DNAs of G puromycin-resistant TT2F cellswere digested with restriction enzyme EcoR I (TAKARA BIO INC., Japan),and then separated on agarose gel electrophoresis. Subsequently,Southern blot was performed, and then homologous recombinants weredetected using the probe shown in the above Example 3. As a result, 0out of 80 clones (0%) were homologous recombinants in thespermidine-free case, and 5 out of 56 lines (9%) were homologousrecombinants in the spermidine-added case.

Example 10 Insertion of Human FGF23 Gene into Knock-in Vector

The following DNAs were synthesized from the nucleotide sequenceencoding a human FGF23 (fibroblast growth factor 23) gene. (SEQ ID NO:13) OST311XM: ATCTCGAGCCACCATGTTGGGGGCCCGCCTCAGG (SEQ ID NO: 14)OST311HX: ATCTCGAGCTAATGATGATGATGATGATGGATGAACTTG GCGAAGGG

Human FGF23 cDNA was amplified in a manner similar to that in the aboveExample 2 using as a template a pcDNA vector containing the full-lengthFGF23 cDNA described in Shimada et al. (Proc. Natl. Acad. Sci. USA, 98:6500-6505, 2000). The PCR reaction solution was subjected to 0.8%agarose gel electrophoresis to separate PCR amplification fragments, andthen target DNA fragments were collected using a QIAquick Gel ExtractionKit (QIAGEN, Germany). The obtained DNA fragments were treated with XhoI at 37° C. for 3 hours, and then collected using a QIAquick PCRPurification Kit (QIAGEN, Germany). pBluescript II SK(−) (TOYOBO, Japan)was treated with Xho I, and then with CIAP (TAKARA BIO INC., Japan) toobtain a pBlueXhoICIAP plasmid. 42 ng of the pBlueXhoICIAP plasmid, 108ng of FGF23 cDNA PCR amplification fragment obtained by treatment withXho I as described above, and 10 μL of DNA Ligation Kit Ver. 2, SolutionI (TAKARA BIO INC., Japan) were mixed, incubated at 16° C. for 30minutes, and then transformed into Escherichia coli DH5α. Plasmid DNA(pBlueFGF23) was prepared from the obtained transformant, and then thenucleotide sequence was confirmed. 9.6 μg of pBlueFGF23 was digestedwith Xho I at 37° C. for 4 hours. FGF23 fragments were collected by 0.8%agarose gel electrophoresis using a QIAquick Gel Extraction Kit (QIAGEN,Germany). The collected FGF23 fragments were separated on 0.8% agarosegel and collected again, thereby preparing a human FGF23 gene forinsertion into pKIκ.

40 ng of dephosphorylated vector, pKIκ-SalI-CIAP (Example 2), 4 ng ofthe above human FGF23 gene for insertion into the knock-in vector, and1.8 μL of DNA Ligation Kit Ver. 2, Solution I (TAKARA BIO INC., Japan)were mixed, incubated at 16° C. for 18 hours, and then transformed intoEscherichia coli XL10-Gold Ultracompetent Cells (TOYOBO, Japan). DNA wasprepared from the obtained transformant, and then the nucleotidesequence was confirmed. A human FGF23 gene-inserted knock-in vector wasprepared using a QIAfilter Plasmid Kit (QIAGEN, Germany). 150 μg of thehuman FGF23 gene-inserted knock-in vector was digested with Xho I (37°C., 6.5 hours) using each of a spermidine-added (1 mM, pH 7.0, SIGMA,U.S.A.) buffer (Roche Diagnostics, Japan, H buffer for restrictionenzyme), and a spermidine-free buffer (described above). Afterphenol/chloroform extraction, 2.5 volumes of 100% ethanol and 0.1 volumeof 3M sodium acetate were added, and then the solution was stored at−20° C. for 16 hours. The vectors linearized with Xho I were collectedby centrifugation, and then sterilized by the addition of 70% ethanol.70% ethanol was removed in a clean bench, and then the product wasair-dried for 1 hour. HBS solution was added to result in a 0.5 μg/μLDNA solution, and the resultant was stored at room temperature for 1hour, thereby preparing a human FGF23 gene-inserted knock-in vector forelectroporation.

Example 11 Preparation of FGF23 Knock-in ES Cell Clone

To obtain a mouse ES cell clone wherein human FGF23-cDNA was inserted byhomologous recombination downstream of an immunoglobulin κ light chaingene, 2 types (spermidine-added and spermidine-free) of the human FGF23knock-in vectors for electroporation prepared in the above Example 10were transfected into a mouse ES cell TT2F (Yagi et al., AnalyticalBiochem., 214:70, 1993) according to an established method (ShinichiAizawa, Bio Manual Series 8, Gene Targeting, YODOSHA, 1995). The methodfor culturing TT2F was performed according to the above-described method(Shinichi Aizawa, supra). Feeder cells used herein were G418-resistantprimary cultured cells (purchased from Invitrogen, Japan) treated withmitomycin C (SIGMA, U.S.A.). First, the propagated TT2F cells weretrypsinized, and then suspended in HBS to achieve 3×10⁷ cells/ml. 0.5 mlof the cell suspension was mixed with 10 μg of the vector DNA, and thenthe mixture was subjected to electroporation using a Gene Pulser Cuvette(electrode distance: 0.4 cm, Bio-Rad, U.S.A.) (capacity: 960 μF,voltage: 240 V, room temperature). The cells that had been subjected toelectroporation were suspended in 10 ml of an ES medium, and then platedon a single 100 mm plastic dish for tissue culture (Falcon, BectonDickinson, U.S.A.) onto which feeder cells had been previouslyinoculated. 36 hours later, the medium was changed with an ES mediumcontaining 0.8 μg/ml puromycin (SIGMA, U.S.A.). Among colonies that hadappeared 7 days later, 80 colonies (spermidine-free) and 56(spermidine-added) colonies were picked up in total, and each colony wasallowed to grow to reach confluence in a 24-well plate. Two-thirds ofthe colonies were suspended in 0.2 ml of a medium for storage (ESmedia+10% DMSO, SIGMA, U.S.A.), and then stored at −80° C. The remainingone-third of the colonies were inoculated onto a 12-well gelatine-coatedplate, and then cultured for 2 days. Then, genomic DNA was prepared from10⁶-10⁷ cells using Puregene DNA Isolation Kits (Gentra Systems,U.S.A.). These puromycin-resistant TT2F cell genomic DNAs were digestedwith a restriction enzyme EcoR I (TAKARA BIO INC., Japan), and thenseparated by agarose gel electrophoresis. Subsequently, Southern blotwas performed, and then homologous recombinants were detected using theprobe shown in the above Example 3. As a result, 0 out of 80 clones (0%)were homologous recombinants in the spermidine-free case, and 3 out of40 lines (8%) were homologous recombinants in the spermidine-added case.The above results show that the addition of spermidine upon preparationof the vector DNA has an effect of increasing the ratio of homologousrecombinants to clones with random insertion.

Example 12 Preparation of Cκ Chain Knock-out Vector pΔCκNeo

pSTneoB (Katoh et al., Cell Struct. Funct., 12: 575, 1987, JapaneseCollection of Research Biologicals (JCRB); Deposit Number: VE039) wasdigested with restriction enzymes Xho I and Sca I, and then fragmentscontaining neo-resistance gene was separated on 0.8% agarose gelelectrophoresis. Target fragments were collected using GENECLEAN II Kit(Qbiogene, Inc., U.S.A.), and then designated as neo fragment. The aboveneo fragment was inserted into the pΔCκSal prepared in the above Example1(2) that had been digested with Sal I, and then dephosphorylated withEscherichia coli alkaline phosphatase. The pΔCκSal was then transformedinto Escherichia coli DH5α. DNA was prepared from the obtainedtransformant, and then the nucleotide sequence of the ligation portionwas confirmed, thereby obtaining a plasmid pΔCκNeo. The structure ofpΔCκNeo is shown in FIG. 2.

Example 13 Preparation of ESΔΔκneo cell clone

In a manner similar to the methods described in the above Examples 2 and3, a mouse ES (TT2F) cell was obtained wherein an allele on the one sideof an endogenous Igκ gene was disrupted by the pΔCκNeo vector (Example12). Since the Igκ genomic homologous region within the pΔCκNeo vectorwas derived from C57BL/6 strain, this vector is inserted preferentiallyinto allele from C57BL/6 in the TT2F cell (derived from F1 of C57BL/6and CBA strains). Using this ES cell clone (ESΔκneo) and the antibodylight chain targeting vector in the WO 00/10383 pamphlet (see Example76), and the method described in the same, a mouse ES cell(ESΔΔκneo/puro) was obtained wherein a further endogenous Igκ geneallele (CBA) was disrupted. For the ESΔΔκneo/puro clone, the puro genewas removed by Cre recombinase according to the method described in theWO 00/10383 pamphlet (see Example 78). Finally, a G418-resistance andpuromycin-sensitive ES cell clone (ESΔΔκneo) was obtained whereinendogenous Igκ genes were disrupted on both alleles. An ES cell clonewas obtained using ESΔΔκneo instead of the TT2F cell clone in theestablishment of the knock-in ES cell clone described in the aboveExample 3. Since the homologous region in the Igκ genome within theknock-in vector was derived from the C57BL/6 strain, in the obtainedhomologous recombinant, this vector is substituted preferentially with aG418-resistance gene in the C57BL/6 allele wherein the G418-resistancegene is present. Hence, the homologous recombinants can be convenientlyscreened for based on sensitivity to G418. In addition, CBA allele ofthe endogenous Igκ gene had been already disrupted in the obtainedhomologous recombinant, so that the Igκ gene of the C57BL/6 allele intowhich a foreign gene had been inserted by the knock-in vector wasexpressed exclusively.

Example 14 Preparation of Type I Knock-in Vectors pKI-I-1 and pKI-I-2

(1) Preparation of Fragments in the Vicinity of Cloning Site

SV40 PolyA signal, Igκ promoter region I or 2, and the multi-cloningsite (MCS) of a restriction enzyme recognition sequence for theinsertion of a target gene were inserted between the termination codonportion and a polyadenylation signal (PolyA signal) of a mouseimmunoglobulin κ chain (Igκ) gene and a puromycin-resistance gene wasinserted downstream of the polyA signal to prepare a DNA fragment. Themethod is specifically shown below.

(1.1) Preparation of Fragment 1 Located Upstream of Cloning Site

The following DNAs were synthesized from the nucleotide sequenceencoding an SV40 polyA signal region obtained from GenBank (NCBI,U.S.A.). (SEQ ID NO: 15) SV GGAATTCAGACATGATAAGATACATTGATGAGTTTGGACAAApoly5: (SEQ ID NO: 16) SV CCCAAGCTTTAATCAGCCATACCACATTTGTAGAGGTTTTACTTpoly3:

An EcoR I recognition sequence was added to the terminus of SVpoly5 thatwas the 5′ primer, and a Hind III recognition sequence was added to theterminus of SVpoly3 that was the 3′ primer. A reaction solution wasprepared using Takara LA-Taq (TAKARA BIO INC., Japan) according to theinstructions by the manufacturer. 10 pmol of each primer and 10 ng ofpSTneoB as a template were added to 50 μl of the reaction solution.After being kept at 94° C. for 1 minute, the solution was subjected to25 cycles of amplification, each cycle consisting of 94° C. for 30seconds and 68° C. for 30 seconds. The obtained reaction solution wassubjected to phenol/chloroform extraction, ethanol precipitation, andthen digestion with EcoR I and Hind III. DNA fragments were separated on0.8% agarose gel electrophoresis, target fragments were collected as anamplification fragments 1 using GENECLEAN II Kit (Qbiogene, Inc.,U.S.A.).

(1.2) Preparation of Fragments 2 and 3 Located Upstream of Cloning Site

The following DNAs were synthesized from the nucleotide sequenceencoding a mouse Igκ promoter region genes obtained from GenBank (NCBI,U. S. A.). (SEQ ID NO: 17) CκpromCCCAAGCTTGAATTAAACAGTTTCAGGGCACATGAAATACTG 1-5: AG (SEQ ID NO: 18)Cκprom GCTCTAGATTTGTCTTTGAATTTTGGTCCCTAGCTAATTACTG 1-3: (SEQ ID NO: 19)Cκprom CCCAAGCTTTGGTGATTATTCAGAGTAGTTTTAGATGAGTGCAT 2-5: (SEQ ID NO: 20)Cκprom GCTCTAGATTTGTCTTTGAACTTTGGTCCCTAGCTAATTACTA 2-3:

A Hind III recognition sequence was added to Cκprom1-5 and Cκprom2-5,which were the 5′ primers, and an Xba I recognition sequence was addedto Cκprom1-3 and Cκprom2-3, which were the 3′ primers. DNA fragments 2and 3 were obtained by using a mouse genomic DNA as a template with aprimer set (Cκprom1-5 and Cκprom1-3) and a primer set (Cκprom2-5 andCκprom2-3) using Takara LA-Taq (TAKARA BIO INC., Japan).

(1.3) Preparation of Multi-Cloning Site Fragment 4

The following DNA fragments were synthesized to prepare a multi-cloningsite fragment containing Xba I, Sal I, Not I, Fse I, Asc I, and Spe I.(SEQ ID NO: 21) MCS (Xba I/ GCTCTAGAGTCGACGCGGCCGCGGCCGGCCGGCGCGC SpeI): CACTAGTC (SEQ ID NO: 22) MCS (Spe I/GACTAGTGGCGCGCCGGCCGGCCGCGGCCGCGTCGACT Xba I): CTAGAGC

The above 2 DNA fragments were annealed to obtain a DNA fragment 4.

(1.4) Preparation of Fragment 5 Located Downstream of Cloning Site

The following DNAs were synthesized from the nucleotide sequenceencoding a mouse Igκ gene obtained from GenBank (NCBI, U.S.A.).CκpolyP5: (SEQ ID NO: 23) GACTAGTAGACAAAGGTCCTGAGACGCCACCACCAGCTCCCCCκpolyP3: (SEQ ID NO: 24) GAAGATCTCAAGTGCAAAGACTCACTTTATTGAATATTTTCTG

A Spe I recognition sequence was added to the terminus of CκpolyP5,which was the 5′ primer, and a Bgl II recognition sequence was added tothe terminus of CκpolyP3 which was the 3′ primer. A DNA fragment 5 wasobtained by using a mouse genomic DNA as a template with the aboveprimers CκpolyP5 and CκpolyP3 using Takara LA-Taq (TAKARA BIO INC.,Japan).

(1.5) Preparation of DNA Fragment I or II Containing DNA Fragments 1, 2,4, and 5, or 1, 3, 4, and 5

Each of the above DNA fragments was treated with restriction enzymes tocleave the recognition sequences added to the 5′ and 3′ sides, and thensubjected to phenol/chloroform extraction, and thenethanol-precipitation. The resulting samples were dissolved in TE. DNAfragments 1, 2, 4, and 5, or 1, 3, 4, and 5 treated with restrictionenzymes were respectively bound by a ligation reaction. The reactionproducts were collected by ethanol precipitation, thereby preparing aDNA fragment I or II.

(2) Construction of Type I Knock-in Vectors, pKI-I-1 and pKI-1-2

(2.1) Preparation of pIgCκΔIRES Plasmid

The pIgCκABP IRES described in the above Example 1 was treated withrestriction enzymes EcoR I and Bgl 11. DNA fragments whereinloxP-puromycin-resistance gene fragments had not been cleaved with Bgl11 were separated and collected by agarose gel electrophoresis, therebyobtaining plasmid pIgCκΔIRES fragment.

(2.2) Preparation of pIgCκΔIRES ProI or pIgCκΔIRES ProII

DNA fragment I was inserted into pIgCκΔIRES to prepare pIgCκΔ IRES ProI,and DNA fragment II was inserted into pIgCκΔIRES to prepare pIgCκΔIRESProII.

(2.3) Preparation of Type I Knock-in Vectors, pKI-I-1 and pKI-1-2

pIgCκΔIRES ProI and pIgCκΔIRES ProII were treated with a restrictionenzyme Xho 1. DNA fragments containing the multi-cloning sites wereseparated and collected by agarose gel electrophoresis. The thusobtained DNA fragments were inserted into pΔCκSal (Example 1) that hadbeen digested with Sal I, and dephosphorylated with Escherichia colialkaline phosphatase, and then the plasmid was transformed intoEscherichia coli DH5α. DNA was prepared from the obtained transformant,the nucleotide sequence of the ligation portion was confirmed, therebyobtaining plasmids pKI-I-1 and pKI-I-2. The structure of pKI-I-1 andthat of pKI-I-2 are shown in FIGS. 3 and 4. The outline of the vectorstructure is as follows.

Specifically, the vector comprises, from the 5′ side, mouse Igκ genomicDNA containing the enhancer sequence downstream of a J fragment and Igκlight chain C region (EcoR I to EcoR I, approximately 5.3 Kb), SV40polyA signal region (approximately 0.4 Kb), a region containing a mouseIgκ promoter 1 (approximately 0.2 Kb; in the case of pKI-I-1, and in thecase of pKI-I-2, mouse Igκ promoter 2), a region containing a multiplecloning site (MCS; approximately 0.02 Kb), a mouse Cκ polyA signalregion (approximately 0.4 Kb), a puromycin-resistance gene cassette(approximately 1.8 Kb), a mouse Igκ genomic DNA (approximately 7.7 Kb)located downstream of a mouse Cκ polyA signal region, a diphtheria toxinA (DT-A) gene cassette (approximately 1.0 Kb), and pUC18 DNA(approximately 2.7 Kb). A knock-in ES cell is prepared by similarlyconducting the procedures in the above Example 2 and the followingexamples by using the above knock-in vector. In a knock-in ES cell, theSV40 polyA signal region was located immediately following thetermination codon of the Cκ structural gene of the mouse Igκ, the mouseIgκ promoter 1 (or 2) and a desired structural gene were locatedimmediately following the SV40 polyA signal region, and a mouse Cκ polyAsignal region was located immediately following the desired structuralgene. By locating the structural gene immediately following the mouseIgκ promoter, it is predicted that the desired gene will be expressed athigh levels equivalent to or greater than that in the case of the pKIκvector using an IRES (Mizuguchi et al., Molecular Therapy Vol.1, No.4,2000, 376-382.). Knock-in ES cells were prepared by conducting similarlythe procedures in the above Example 2 and the following examples usingthe knock-in vector obtained as described above instead of using pKIκ inthe above Example 1.

Example 15 Construction of Type II Knock-in Vectors, pKI-II-1 andpKI-II-2

(1) Preparation of Fragment in the Vicinity of Cloning Site

A restriction enzyme recognition sequence multi-cloning site (MCS) forthe insertion of a Cκ PolyA signal, a Cκ promoter region 1 or 2, and atarget gene was introduced between the termination codon portion and theCκ polyadenylation signal (PolyA signal) of a mouse immunoglobulin κchain (Igκ) gene, and a puromycin-resistance gene was introduceddownstream of Cκ polyA signal, thereby preparing a genomic fragment. Thepreparation method is specifically described as follows.

(1.1) Preparation of Fragment 6 Located Upstream of Cloning Site

The following DNAs were synthesized from the nucleotide sequence of CκpolyA signal region obtained from GenBank (NCBI, U.S.A.). CκpolyT5: (SEQID NO: 25) GGAATTCAGACAAAGGTCCTGAGACGCCACCACCAGCTCCCC CκpolyT3: (SEQ IDNO: 26) CCCAAGCTTGCCTCCTCAAACCTACCATGGCCCAGAGAAATAAG

An EcoR I recognition sequence was added to the terminus of CκpolyT5which was the 5′ primer, and an Hind III recognition sequence was addedto the terminus of CκpolyT3, which was the 3′ primer. A reactionsolution was prepared using Takara LA-Taq (TAKARA BIO INC., Japan)according to the instructions by the manufacturer. To 50 μL of thereaction solution, 10 pmol of each primer and 10 ng of mouse genomic DNAas a template were added. The solution was kept at 94° C. for 1 minute,and then subjected to 25 cycles of amplification, each cycle consistingof 94° C. for 30 seconds and 68° C. for 30 seconds. The obtainedreaction solution was subjected to phenol/chloroform extraction, ethanolprecipitation, and then digestion with EcoR I and Hind III. DNAfragments were separated on 0.8% agarose gel electrophoresis, targetfragments were collected using GENECLEAN II Kit (Qbiogene, Inc., U.S.A.)to obtain an amplification fragment 6.

(1.2) Preparation of DNA Fragment III or IV Containing DNA Fragments 6,2, 4, and 5 or 6, 3, 4, and 5

Each of the above DNA fragments was treated with restriction enzymes tocleave the recognition sequences added to the 5′ and 3′ sides,respectively, and then subjected to phenol/chloroform extraction, andthen ethanol-precipitation. The resulting samples were dissolved in TE.DNA fragments 6, 2, 4, and 5, or 6, 3, 4, and 5 treated with restrictionenzymes, were bound by ligation reactions, respectively. The reactionproducts were collected by ethanol precipitation, thereby preparing aDNA fragment III or IV.

(2) Construction of Type II Knock-in Vectors, pKI-II-1 and pKI-II-2

(2.1) Preparation of pIgCκA IRES ProIII or pIgCκΔIRES ProIV

DNA fragment III was inserted into pIgCκΔIRES, so as to preparepIgCκΔIRES ProIII, and DNA fragment IV was inserted into pIgCκΔIRES, soas to prepare pIgCκΔIRES ProIV.

(2.2) Preparation of Type II Knock-in Vectors, pKI-II-1 and pKII-I-2

pIgCκΔIRES ProIII and pIgCκΔIRES ProIV were treated with a restrictionenzyme Xho I. DNA fragments containing the multi-cloning site wereseparated and collected by agarose gel electrophoresis. The thusobtained DNA fragment was inserted into pCκSal that had been digestedwith Sal I and dephosphorylated with Escherichia coli alkalinephosphatase. Then the plasmid was transformed into Escherichia coliDH5κ. DNA was prepared from the obtained transformant and the nucleotidesequence of the ligation portion was confirmed, thereby obtainingplasmids pKI-II-1 and pKI-II-2. The structure of pKI-II-1 and that ofpKI-II-2 are shown in FIGS. 5 and 6. The outline of the vector structureis as follows.

Specifically, the vector comprises, from the 5′ side, mouse Igκ genomicDNA containing the enhancer sequence downstream of a J fragment and theIgκ light chain C region (EcoR I to EcoR I, approximately 5.3 Kb), aregion containing mouse Cκ polyA (approximately 0.4 Kb), a regioncontaining a mouse Igκ promoter 1 (approximately 0.2 Kb; in the case ofpKI-II-1, and in the case of pKl-II-2, mouse Igκ promoter 2), a regioncontaining the multiple cloning site (MCS; approximately 0.02 Kb), aregion containing mouse Cκ polyA (approximately 0.4 Kb), apuromycin-resistance gene cassette (approximately 1.8 Kb), a mouse Igκgenomic DNA located downstream of the mouse Cκ polyA (approximately 7.7Kb), a diphtheria toxin A (DT-A) gene cassette (approximately 1.0 Kb),and pUC18 DNA (approximately 2.7 Kb). A knock-in ES cell is prepared bysimilarly conducting the procedures in the above Example 2 and thefollowing examples by using the above knock-in vector. In the knock-inES cell, the mouse Cκ polyA signal region was located immediatelyfollowing the termination codon of the Cκ structural gene of the mouseIgκ, the mouse Igκ promoter 1 (or 2) and a desired structural gene werelocated immediately following the mouse Cκ polyA signal region, and amouse Cκ polyA signal region was located immediately following thedesired structural gene. By locating the structural gene immediatelyfollowing the mouse Igκ promoter, it is predicted that the desired genewill be expressed at high levels equivalent to, or greater than that inthe case of the vector pKIκ using an IRES (Mizuguchi et al., MolecularTherapy Vol.1, No.4, 2000, 376-382.). Knock-in ES cells were prepared byconducting similarly the procedures in the above Example 2 and thefollowing examples using the knock-in vector obtained as described aboveinstead of using pKIκ in the above Example 1.

Example 16 High Level Expression of Foreign Gene in Muscle TissueUtilizing a Myoglobin Gene Regulatory Sequence

Myoglobin is an oxygen-binding heme protein that is present in largequantities in muscle, and the expression thereof is limited to themuscle tissue such as skeletal muscle or the heart (Tomizuka et al.,Nature Genet. 16: 133-43, 1997). As described above, by the injection ofa mouse ES cell, which contains a foreign gene located so as to beexpressed under the regulation of the regulatory sequence of a myoglobingene, into an embryo derived from a mouse strain deficient in theability to form muscle tissue (e.g., a myogenin gene-deficienthomozygote, Nabeshima et al., Nature 364: 532-5, 1993), the foreign genecan be expressed at high levels in chimeric mice and offsprings thereof.

For example, a target gene fragment wherein a Kozak sequence is locatedimmediately upstream of the initiation codon can be inserted byhomologous recombination into an exon containing the initiation codon ofa myoglobin gene. In this case, the myoglobin gene of an allele havingthe target gene inserted therein is not expressed normally. However,since the myoglobin-gene-knock-out mouse is normal (Garry et al.,Nature, 39: 5905-8, 1998), it is thought that non-expression of oneallele of this gene does not have any effect on the phenotype of thechimeric mouse. Furthermore, in a manner similar to that in the aboveExample 1, IRES+Kozak sequence+target gene can be inserted within the 3′untranslated sequence located downstream of the termination codon of amyoglobin gene. In this case, a myoglobin gene of an allele having atarget gene inserted therein can also be expressed.

A chimeric mouse can be produced by injecting ES cells altered asdescribed above into an 8-cell-stage embryo obtained by the crossing ofheterozygotes of a myogenin gene-disrupted mouse strain (Nabeshima etal., supra). There is a 25% probability of embryo being a homozygote(that is, deficient in the ability to form muscle tissue). Thus, in 25%of the chimeric mice that are born, muscle tissues are mainly derivedfrom knock-in ES cells. Moreover, in the muscle cells of a mouse thathave differentiated from knock-in ES cells, a foreign gene is expressedat high levels under the control of the regulatory sequence of amyoglobin gene.

Example 17 Construction of Mouse FGF23 Knock-out Vector

(1) Preparation of Mouse FGF23 Genomic Region Fragment

The following DNAs (primers) were synthesized from the nucleotidesequence encoding a mouse FGF23 (fibroblast growth factor 23) geneobtained from GenBank (NCBI, U.S.A.). P51: GACTCCTGGTGGGCGTGCTC (SEQ IDNO: 27) P31: GGTGCCATCTACATGACCAT (SEQ ID NO: 28)

A reaction solution was prepared using Takara EX-Taq (TAKARA BIO INC.,Japan) according to the instructions by the manufacturer. To 50 μL ofthe reaction solution, 10 pmol of each of the above primers and 25 ng ofmouse TT2F cell-derived genomic DNA as a template were added. Thesolution was kept at 94° C. for 2 minutes, and then subjected to 35cycles of amplification, each cycle consisting of 94° C. for 30 seconds,60° C. for 30 seconds, and 72° C. for 20 seconds. PCR amplificationfragments of 173 mer were collected from the obtained reaction solutionby 2% agarose gel electrophoresis. Amplification fragments werecollected from the excised gel using a QIAquick Gel Extraction Kit(QIAGEN, Germany) according to the instructions by the manufacturer.Thus, an FGF23P probe used for the selection of BAC clones containingthe FGF23 genomic region was obtained.

(2) Selection of BAC Clones Containing Mouse FGF23 Genomic Region

BAC clones containing the FGF23 genomic region were screened by using ahigh density filter: BAC mouse C57/BL6 (KURABO, Japan) and the obtainedFGF23P as a probe. As a result, 3 positive clones were obtained. Theclone ID/address of these 3 types of clones were 17d5, 235I6, and211k15. BAC clones were obtained based on the information on thesepositive clone numbers.

It was confirmed whether or not the obtained BAC clones containing thetarget FGF23 genomic region. To briefly explain this, a reactionsolution was prepared using Takara EX Taq (TAKARA BIO INC., Japan)according to the instructions by the manufacturer. To 50 μL of thereaction solution, 10 pmol of each of P51 and P31 primers, and 100 ng ofBAC clone DNA as a template were added. The solution was kept at 94° C.for 2 minutes, and then subjected to 30 cycles of amplification, eachcycle consisting of 94° C. for 30 seconds, 60° C. for 30 seconds, and72° C. for 20 seconds. The obtained reaction solution was analyzed by 2%agarose gel electrophoresis, a band of 173 mer was detected in case ofclone numbers 235I6 and 211K15 had been used as templates. According tothis result, it was confirmed that BAC clones of clone numbers 235I6 and211K15 contained the target FGF23 genomic region.

(3) Subcloning of BAC Clone to pBluescript II SK(−)

0.05 U of Sau3AI (Roche Diagnostics K. K., Japan) was added to 2.5 μg ofDNA prepared from BAC clone of clone number 211K15, and the resultantwas incubated at 37° C. for 20 minutes. After separation on 0.8% gel, aband with a molecular weight of approximately 15 to 7 Kb was excised,and then DNA fragments were collected using a QIAquick Gel ExtractionKit (QIAGEN, Germany) according to the instructions by the manufacturer.

The above collected DNA fragments were inserted into pBluescript IISK(−) (TOYOBO, Japan) that had been digested with BamH I, and thendephosphorylated with Escherichia coli alkaline phosphatase. Theresultant plasmid was then transfected into Escherichia coli MAXEfficiency STBL2 Competent Cells (Invitrogen). Clones wherein the DNAfragment had been inserted into the plasmid were selected using a platesupplemented with IPTG/X-gal, so that 100 white colonies were collected.

(4) Selection of Clones Containing FGF23 Genomic Region

A reaction solution was prepared using Takara EX Taq (TAKARA BIO INC.,Japan) according to the instructions. To 50 μL of the reaction solution,10 pmol of each of P51 and P31 primers and 25 ng of plasmid DNA as atemplate were added. The solution was kept at 94° C. for 2 minutes, andthen subjected to 35 cycles of amplification, each cycle consisting of94° C. for 30 seconds, 60° C. for 30 seconds, and 72° C. for 20 seconds.Clones for which an amplification fragment of 173 mer was detected using2% agarose gel electrophoresis were selected, thereby obtaining clonenumbers 33 and 94.

(5) Selection of Clone Containing a Region of Approximately 5 Kb Located5′ Upstream, and a Region of Approximately 2 Kb Located 3′ Downstream,of FGF23 Exon 1 Region

KO53 and KO35 were synthesized from the nucleotide sequence of mouseFGF23 gene obtained from GenBank (NCBI, U.S.A.), and KO55 and KO33 weresynthesized from the nucleotide sequence of pBluescript II SK (−). KO55:AATTAACCCTCACTAAAGGGAA (SEQ ID NO: 29) KO53: CAAGCAATGGGGAAGTGTCTGG (SEQID NO: 30) KO35: CGGCTACAGCCAGGACCAGCTA (SEQ ID NO: 31) KO33:GTAATACGACTCACTATAGGGCGA (SEQ ID NO: 32)

A reaction solution was prepared using KOD-Plus—(TOYOBO, Japan)according to the instructions by the manufacturer. To 50 μL of thereaction solution, in case of the target 5′ region was selected, 10 pmoleach of KO55 and KO53 primers, or in case of the target 3′ region wasselected, 10 pmol each of KO35 and KO33 primers, and 25 ng of plasmidDNA derived from clone number 33 or 94 as a template were added. Thesolution was kept at 94° C. for 2 minutes, and then subjected to 30cycles of amplification, each cycle consisting of 94° C. for 15 seconds,65° C. for 20 seconds, and 68° C. for 10 minutes. The size of theobtained amplification fragments were analyzed with 0.8% agarose gel. Incase of the DNA prepared from clone number 33 was used as a template,approximately 5 Kb of a PCR amplification fragment was detected with aprimer set for the selection of the 5′ region, and approximately 2 Kb ofa PCR amplification fragment was detected with the primer set for theselection of the 3′ region. These results revealed that the targetgenomic region fragment had been subcloned into the clone of clonenumber 33.

(6) Preparation of PCR Amplification Fragment 5′ KO Having RestrictionEnzyme Sites at Both Termini of 5′ Genomic Homologous Region

Using DNA derived from clone number 33, each nucleotide sequence ofapproximately 700 bp at both ends of the 5′ upstream region of subclonedFGF23 exon 1 was determined. Based on the obtained information,approximately 5 Kb of 5′ upstream region of FGF23 exon 1 was amplified,and a PCR primer set of NotI55 and FseI53, for the addition of the Not Isite to the 5′ side and the Fse I site to the 3′ side of theamplification fragment, were designed. NotI55: (SEQ ID NO: 33)ATAAGAATGCGGCCGCTAAACTATAGCATCCACTGGGAATC AACATCTGAGACATCCTA FseI53:(SEQ ID NO: 34) CGGGCCGGCCCGCGGGACTTTTAAAGGGTGGTGGTGTGAC ATCAAGC

A reaction solution was prepared using KOD-Plus—(TOYOBO, Japan)according to the instructions by the manufacturer. To 50 μL of thereaction solution, 10 pmol each of the above 2 primers and 25 ng ofplasmid DNA of clone number 33 as a template were added. The solutionwas kept at 94° C. for 2 minutes, and then subjected to 25 cycles ofamplification, each cycle consisting of 94° C. for 15 seconds and 68° C.for 10 minutes. The thus obtained amplification fragments ofapproximately 5 Kb were separated and recovered using 0.8% agarose gel.Amplification fragments were collected from the collected gel using aQIAquick Gel Extraction Kit (QIAGEN, Germany) according to theinstructions by the manufacturer. The collected PCR amplificationfragments were digested with enzymes Not I and Fse I, and then separatedand collected using 0.8% agarose gel. Enzyme-treated fragments wererecovered from the collected gel using a QIAquick Gel Extraction Kit(QIAGEN, Germany) according to the instructions by the manufacturer.

(7) Preparation of PCR Amplification Fragment 3′KO Having RestrictionEnzyme Sites at Both Termini of the 3′ Genomic Homologous Region

Using DNA derived from clone number 33, each nucleotide sequence ofapproximately 700 bp at both ends of the 3′ downstream region ofsubcloned FGF23 exon 1 was determined. Based on the obtainedinformation, approximately 1.8 Kb of 3′ downstream region of FGF23 exon1 was amplified, and a PCR primer set of AscI55 and XhoI53, for theaddition of the Asc I site to the 5′ side and the Xho I site to the 3′side of the amplification fragment, were designed. AscI55: (SEQ ID NO:35) GGCGCGCCCACTGCTAGAGCCTATCCAGACACTTCCCCATTGC XhoI53: (SEQ ID NO: 36)CCGCTCGAGCGGTGTTCCAGACTGACCACCTTTCAACAAAGAGATTC

A reaction solution was prepared using KOD-Plus—(TOYOBO, Japan)according to the instructions by the manufacturer. To 50 μL of thereaction solution, 10 pmol each of the above 2 primers and 25 ng ofplasmid DNA of clone number 33 as a template were added. The solutionwas kept at 94° C. for 2 minutes, and then subjected to 25 cycles ofamplification, each cycle consisting of 94° C. for 15 seconds and 68° C.for 10 minutes. The thus obtained amplification fragments withapproximately 1.8 Kb were separated and collected using 0.8% agarosegel. Amplification fragments were recovered from the collected gel usinga QIAquick Gel Extraction Kit (QIAGEN, Germany) according to theinstructions by the manufacturer. The collected PCR amplificationfragments were digested with restriction enzymes Asc I and Xho I, andthen separated and collected using 0.8% agarose gel. Enzyme-treatedfragments were recovered from the collected gel using QIAquick GelExtraction Kit (QIAGEN, Germany) according to the instructions by themanufacturer.

(8) Preparation of Cassette Vector pBlueLAB-LoxP-Neo-DT-A

The following DNAs were synthesized to add new restriction enzyme sitesto a vector. LinkA1: TCGAGTCGCGACACCGGCGGGCGCGCCC (SEQ ID NO: 37)LinkA2: TCGAGGGCGCGCCCGCCGGTGTCGCGAC (SEQ ID NO: 38) LinkB1:GGCCGCTTAATTAAGGCCGGCCGTCGACG (SEQ ID NO: 39) LinkB2:AATTCGTCGACGGCCGGCCTTAATTAAGC (SEQ ID NO: 40)

A reaction solution wherein pBluescript II SK(−) (TOYOBO, Japan) hadbeen treated with Sal I and Xho I restriction enzymes was subjected tophenol/chloroform extraction, and ethanol precipitation. To add the newrestriction enzyme sites Nru I, SgrA I and Asc I, to the plasmid, LinkA1and LinkA2 were synthesized. A linker comprising the two oligo DNAs wasinserted into the plasmid treated with restriction enzymes, and then theplasmid was transformed into Escherichia coli DH5α. DNA was preparedfrom the obtained transformant, so that a plasmid pBlueLA was obtained.

Subsequently, a reaction solution wherein PBlueLA had been treated withrestriction enzymes Not I and EcoR I was subjected to phenol/chloroformextraction, and ethanol precipitation. To add new restriction enzymesites Pac I, Fse I and Sal I to the plasmid, LinkB1 and LinkB2 weresynthesized. A linker comprising the two oligo DNAs was inserted intothe plasmid treated with restriction enzymes, and then the plasmid wastransformed into Escherichia coli DH5α. DNA was prepared from theobtained transformant, so that a plasmid pBlueLAB was obtained.

The pLoxP-STneo plasmid described in the WO 00/10383 pamphlet (supra)was digested with Xho I, thereby obtaining a Neo-resistance gene(LoxP-Neo) having a LoxP sequence at both ends. Both ends of LoxP-Neowere blunt-ended using T4 DNA polymerase, so that LoxP-Neo-B wasobtained.

After the above pBlueLAB was digested with EcoR V, the reaction solutionwas subjected to phenol/chloroform extraction and ethanol precipitation.After LoxP-Neo-B was inserted, the plasmid was transformed intoEscherichia coli DH5α. DNA was prepared from the obtained transformant,so that a plasmid pBlueLAB-LoxP-Neo was obtained.

After pMC1DT-A (Invitrogen) was digested with Xho I and Sal I, theproduct was applied to 0.8% agarose gel. After a band of approximately 1Kb was separated and collected, DT-A fragments were collected using aQIAquick Gel Extraction Kit (QIAGEN, Germany) according to theinstructions by the manufacturer.

After pBlueLAB-LoxP-Neo was digested with Xho I, the reaction solutionwas subjected to phenol/chloroform extraction and ethanol precipitation.After DT-A fragment was inserted, the plasmid was transformed intoEscherichia coli DH5α. DNA was prepared from the obtained transformant,and a cassette vector pBlueLAB-LoxP-Neo-DT-A to be used for thepreparation of a knock-out vector was obtained.

(9) Construction of FGF 23 Knock-out Vector

After pBlueLAB-LoxP-Neo-DT-A was digested with Asc I and Xho I, theobtained DNA fragment of approximately 7.3 Kb was separated and purifiedusing 0.8% agarose gel, and then dephosphorylated with Escherichia colialkaline phosphatase. The genomic fragment 3′ KO prepared in (7) wasinserted into the fragment, and then the fragment was transformed intoEscherichia coli MAX Efficiency STBL2 Competent Cells (Invitrogen). DNAwas prepared from the obtained transformant. The nucleotide sequence ofthe inserted portion of the PCR amplification fragment was compared withthe nucleotide sequence information obtained using clone number 33,thereby confirming that no amplification errors resulting from PCRamplification were contained, and confirming the nucleotide sequence ofthe ligation portion. Thus, a plasmid pBlueLAB-LoxP-Neo-DT-A-3′KO wasobtained.

After pBlueLAB-LoxP-Neo-DT-A-3′KO was digested with Not I and Fse I, theobtained DNA fragment of approximately 9.1 Kb was separated and purifiedusing 0.8% agarose gel, and then dephosphorylated with Escherichia colialkaline phosphatase. The genomic fragment 5′ KO prepared in (6) wasinserted into the fragment, and then the product was transformed intoEscherichia coli MAX Efficiency STBL4 Competent Cells (Invitrogen). DNAwas prepared from the obtained transformant, and then the nucleotidesequence of the inserted portion of the PCR amplification fragment wascompared with a region of approximately 3 Kb that had been obtained fromthe nucleotide sequence information obtained using clone number 33,thereby confirming that no amplification errors resulting from PCRamplification were contained within the range and confirming thenucleotide sequence of the ligation portion. Thus, a plasmidpBlueLAB-LoxP-Neo-DT-A-3′KO-5′KO was obtained. The structure of mouseFGF23 knock-out vector: pBlueLAB-LoxP-Neo-DT-A-3′KO-5′KO is shown inFIG. 7.

(10) Preparation of FGF23 Knock-out Vector for Electroporation

Using a buffer (Roche Diagnostics, Japan, H buffer for restrictionenzyme) supplemented with spermidine (1 mM, pH 7.0, SIGMA U.S.A.), 150μg of pBlueLAB-LoxP-Neo-DT-A-3′KO-5′KO was digested with Not I or Xho Iat 37° C. for 5 hours. After phenol/chloroform extraction, 2.5 volumesof 100% ethanol and 0.1 volume of 3M sodium acetate were added, and thenthe solution was kept at −20° C. for 16 hours. The linearized vectordigested with Not I or Xho I was collected by centrifugation, and thensterilized by the addition of 70% ethanol. 70% ethanol was removed in aclean bench and then air-dried for 1 hour. An HBS solution was added toresult in a 0.5 μg/μL DNA solution and the resultant was stored at roomtemperature for 1 hour, so that FGF23 knock-out vectors forelectroporation, FGF-KO-NotI and FGF-KO-XhoI were prepared. FGF-KO-NotIand FGF-KO-XhoI have termini shown with Not I and Xho I, respectively,in FIG. 7.

(11) Preparation of Probe for Genomic Southern Analysis

The following DNAs for obtaining oligo DNA containing a region of 525mer immediately downstream of the 3′ KO were synthesized based on thenucleotide sequence information using DNA derived from clone number 33.OST3S5: TCAGTCTAAATGGCAGGCTTACAGACATCC (SEQ ID NO: 41) OST3S3:TGAGGCAGATCATTCCATCTTGTCAAGACC (SEQ ID NO: 42)

A reaction solution was prepared using Takara EX Taq (TAKARA BIO INC.,Japan) according to the instructions by the manufacturer. To 50 μL ofthe reaction solution, 10 pmol of each of the above 2 primers and 25 ngof DNA derived from BAC of clone number 211K15 as a template were added.The solution was kept at 94° C. for 2 minutes, and then subjected to 33cycles of amplification, each cycle consisting of 94° C. for 30 seconds,60° C. for 30 seconds, and 72° C. for 1 minute. The obtained 544 mer ofamplification fragments were separated and collected using 0.8% agarosegel. From the collected gel, a 3′ genomic probe for the Southern, 3′KO-prob, was collected using a QIAquick Gel Extraction Kit (QIAGEN,Germany) according to the instructions by the manufacturer.

Example 18 Preparation of FGF23 Knock-out ES Cell Clone

To obtain a mouse FGF23 knock-out ES cell line by homologousrecombination, the pBlueLAB-LoxP-Neo-DT-A-3′KO-5′KO prepared in Example17 was linearized with a restriction enzyme Not I or Xho I (TAKARA BIOINC., Japan), and then transfected into a mouse ES cell TT2F (Yagi etal., Analytical Biochem., 214: 70, 1993) according to the establishedmethod (Shinichi Aizawa, Bio Manual Series 8, Gene Targeting, YODOSHA,1995).

The method for culturing TT2F was performed according to theabove-described method (Shinichi Aizawa, supra). Feeder cells usedherein were G418-resistant primary cultured cells (purchased fromInvitrogen, Japan) treated with mitomycin C (SIGMA, U.S.A.). First, thepropagated TT2F cells were trypsinized, and then suspended in HBS toresult in a 3×1⁷cells/ml suspension. 0.5 ml of the cell suspension wasmixed with 10 μg of the vector DNA, and the resultant was subjected toelectroporation using a Gene Pulser Cuvette (electrode distance: 0.4 cm,Bio-Rad, U.S.A.) (capacity: 960 μF; voltage: 240 V; room temperature).The cells that had been subjected to electroporation were suspended in10 ml of an ES medium, and then plated onto a single I 00 mm plasticdish for tissue culture (Falcon, Becton Dickinson, U.S.A.) onto whichfeeder cells had been previously inoculated. 36 hours later, the mediumwas changed with an ES medium containing 0.8 μg/ml puromycin (SIGMA,U.S.A.). Colonies that had appeared 7 days later were picked up, andeach colony was allowed to grow to reach confluence in a 24-well plate.Two-thirds of the colonies were suspended in 0.2 ml of a medium forstorage (ES medium+10% DMSO, SIGMA, U.S.A.), and then stored at −80° C.The remaining one-third of the colonies were inoculated onto a 12-wellgelatine-coated plate, and then cultured for 2 days. Then, genomic DNAwas prepared from 10⁶-10⁷ cells using Puregene DNA Isolation Kits(Gentra Systems, U.S.A.). These G puromycin-resistant TT2F cell genomicDNAs were digested with a restriction enzyme Hind III (TAKARA BIO INC.,Japan), and then separated on 0.8% agarose gel electrophoresis.Subsequently, Southern blot was performed, and then homologousrecombinants were detected using as a probe a DNA fragment (3′ KO-prob;see Example 17 (1)) located in a downstream region immediately followingthe 3′ homologous region of the knock-out vector. In wild-type TT2Fcells, 2 bands (approximately 6 Kb and approximately 4.5 Kb) weredetected by digestion with Hind III. It was predicted that in thehomologous recombinants, the band of approximately 6 Kb would disappearand instead, a new band of approximately 2.5 Kb would appear. In theneomycin-resistant clone, a new band of approximately 2.5 Kb wasconfirmed. Hence, in these clones, a neomycin-resistance gene (includingthe restriction enzyme sites derived from the knock-out vector at bothof its ends) had been inserted into a region of the initiation codon ofthe FGF23 gene (a range between −142 and +59, when the position of A ofATG corresponding to the initiation methionine is supposed to be +1, anda position located 1 nucleotide upstream from this A is supposed to be−1) of one of the alleles. As a result of Southern analysis, whenpBlueLAB-LoxP-Neo-DT-A-3′KO-5′KO was linearized with a restrictionenzyme Not I, 16 out of 90 clones (18%) were homologous recombinants,and when pBlueLAB-LoxP-Neo-DT-A-3′KO-5′KO was linearized with arestriction enzyme Xho I, 1 out of 28 clones (3.6%) were homologousrecombinants. These results showed that, when a targeting vector islinearized, a case where negative selection marker gene DT-A is notlocated at the terminus of the vector structure is more advantageous inhomologous recombination compared with a case where the same is locatedat the terminus (see also FIG. 7). In cases where 6 different types ofknock-out vectors having structures similar to the above cases were usedfor each different gene, an average homologous recombination efficiencyof 22.3%, which was higher than generally reported homologousrecombination efficiencies (approximately several percent), could beobtained.

All publications, patents, and patent applications cited herein areincorporated herein by reference in their entirety.

INDUSTRIAL APPLICABILITY

According to the present invention, chimeric non-human animals (e.g.,chimeric mice) can be obtained expressing at high levels a desiredprotein efficiently and reliably compared with conventional methods.Since an embryo used as a host embryo in the present invention isdeficient in a cell and/or tissue wherein an introduced gene encoding adesired protein is expressed, all the cells and/or tissues in the thusproduced chimeric non-human animal are all derived from pluripotentcells containing the introduced nucleic acid sequence (structural gene),so that the desired protein can be expressed at a high efficiency. Inthe present invention, preferably the expression system of animmunoglobulin light chain, and particularly preferably the expressionsystem of a κ chain, is utilized. The homologous recombinationefficiency in this Igκ gene locus is 5% or more, which is higher thanthose of conventional methods. Therefore, the present invention can beused for the production of a desired protein by the high-levelexpression of a gene encoding the desired protein, or for the analysisof in vivo functions of a gene or a protein with unknown functions.

SEQUENCE FREE TEXT

-   SEQ ID NOS: 1 to 6: synthetic oligonucleotide-   SEQ ID NOS: 7 and 8: Sal I recognition sequence-   SEQ ID NOS: 9 to 20: synthetic oligonucleotide-   SEQ ID NOS: 21 and 22: multi-cloning site-   SEQ ID NOS: 23 to 26: synthetic oligonucleotide-   SEQ ID NO: 29: synthetic oligonucleotide-   SEQ ID NOS: 32 to 40: synthetic oligonucleotide

1. A method for producing a chimeric non-human animal, which comprisesthe steps of: 1) preparing a pluripotent cell derived from a non-humananimal containing a genome wherein a nucleic acid sequence encoding adesired protein is located so that the expression of the desired proteinis regulated by the regulatory region of a gene that is expressed in atleast a specific cell and/or tissue; 2) obtaining a chimeric embryo byinjecting the pluripotent cell prepared in the step I into a host embryoof a non-human animal strain that is deficient in the specific celland/or tissue; 3) transplanting the chimeric embryo obtained in the step2 to a foster parent non-human animal of the same species; and 4)selecting a chimeric non-human animal expressing the desired protein inat least the specific cell and/or tissue from offsprings obtained afterthe transplantation step 3:
 2. The method of claim 1, wherein thenucleic acid sequence encoding a desired protein is located downstreamof the regulatory region of a gene that is expressed in a specific celland/or tissue.
 3. The method of claim 1, wherein the nucleic acidsequence encoding a desired protein is located downstream of an internalribosomal entry site located downstream of the termination codon of agene that is expressed in a specific cell and/or tissue.
 4. The methodof claim 1, wherein a sequence containing an internal ribosomal entrysite and the nucleic acid sequence encoding a desired protein is locatedbetween the termination codon and a polyA signal region of a gene thatis expressed in a specific cell and/or tissue.
 5. The method of claim 1,wherein a sequence containing a polyA signal region, a promotersequence, and the nucleic acid sequence encoding a desired protein islocated between the termination codon and a polyA signal region of agene that is expressed in a specific cell and/or tissue.
 6. The methodof claim 5, wherein the promoter sequence is derived from a gene that isexpressed in a specific cell and/or tissue.
 7. The method of claim 1,wherein a sequence containing a promoter sequence, the nucleic acidsequence encoding a desired protein, and a polyA signal region islocated downstream of a polyA signal region of a gene that is expressedin a specific cell and/or tissue.
 8. The method of claim 7, wherein thepromoter sequence is derived from a gene that is expressed in a specificcell and/or tissue.
 9. The method of claim 7, wherein the distancebetween the polyA signal region of the gene that is expressed in aspecific cell and/or tissue and the promoter sequence is preferably lessthan 1 Kb.
 10. The method of claim 1, wherein the pluripotent cellcontains both a genome wherein the nucleic acid sequence encoding adesired protein is located so that the expression of the desired proteinis regulated by the regulatory region of a gene that is expressed in atleast a specific cell and/or tissue, and a genome wherein the allele ofthe gene that is expressed in the specific cell and/or tissue isinactivated.
 11. The method of claim 1, wherein the pluripotent cell isan embryonic stem cell.
 12. The method of claim 1, wherein the chimericnon-human animal is selected from the group consisting of mice, cattle,pigs, monkeys, rats, sheep, goats, rabbits, and hamsters.
 13. The methodof claim 1, wherein the chimeric non-human animal is a mouse.
 14. Themethod of claim 1, wherein a combination of a gene that is expressed ina specific cell and/or tissue and the specific cell and/or tissuedeficient in a non-human animal strain is selected from the groupconsisting of the following (1) to (7): (1) an immunoglobulin lightchain or heavy chain gene and a B-lymphocyte; (2) a T-cell receptor geneand a T-lymphocyte; (3) a myoglobin gene and a muscle cell; (4) acrystallin gene and a crystalline lens of an eyeball; (5) a renin geneand a kidney tissue; (6) an albumin gene and a liver tissue; and (7) alipase gene and a pancreas tissue.
 15. The method of claim 1, whereinthe combination of a gene that is expressed in a specific cell and/ortissue and the specific cell and/or tissue deficient in a non-humananimal strain is that of an immunoglobulin light chain κ gene and aB-lymphocyte.
 16. A method for producing a non-human animal expressing adesired protein, which comprises obtaining an offspring capable ofexpressing the desired protein by crossing the chimeric non-human animalproduced by the method of any one of claims 1 to
 15. 17. A chimericnon-human animal, which is derived from a pluripotent cell derived froma non-human animal containing a genome wherein a nucleic acid sequenceencoding a desired protein is located so that the expression of thedesired protein is regulated by the regulatory region of a gene that isexpressed in a specific cell and/or tissue and a host embryo of anon-human animal strain deficient in the specific cell and/or tissue,and is capable of expressing the desired protein in at least thespecific cells and/or tissues.
 18. The chimeric non-human animal ofclaim 17, wherein the nucleic acid sequence encoding a desired proteinis located downstream of the regulatory region of a gene that isexpressed in a specific cell and/or tissue.
 19. The chimeric non-humananimal of claim 17, wherein the nucleic acid sequence encoding a desiredprotein is located downstream of an internal ribosomal entry sitelocated downstream of the termination codon of a gene that is expressedin a specific cell and/or tissue.
 20. The chimeric non-human animal ofclaim 17, wherein a sequence containing an internal ribosomal entry siteand the nucleic acid sequence encoding a desired protein is locatedbetween the termination codon and a polyA signal region of a gene thatis expressed in a specific cell and/or tissue.
 21. The chimericnon-human animal of claim 17, wherein a sequence containing a polyAsignal region, a promoter sequence, and the nucleic acid sequenceencoding a desired protein is located between the termination codon anda polyA signal region of a gene that is expressed in a specific celland/or tissue.
 22. The chimeric non-human animal of claim 21, whereinthe promoter sequence is derived from a gene that is expressed in aspecific cell and/or tissue.
 23. The chimeric non-human animal of claim17, wherein a sequence containing a promoter sequence, the nucleic acidsequence encoding a desired protein, and a polyA signal region islocated downstream of a polyA signal region of a gene that is expressedin a specific cell and/or tissue.
 24. The chimeric non-human animal ofclaim 23, wherein the promoter sequence is derived from a gene that isexpressed in a specific cell and/or tissue.
 25. The chimeric non-humananimal of claim 23, wherein the distance between the polyA signal regionof a gene that is expressed in a specific cell and/or tissue and thepromoter sequence is less than 1 Kb.
 26. The chimeric non-human animalof claim 17, wherein the pluripotent cell contains both a genome whereinthe nucleic acid sequence encoding a desired protein is located so thatthe expression of the desired protein is regulated by the regulatoryregion of a gene that is expressed in at least a specific cell and/ortissue, and a genome wherein the allele of the gene that is expressed inthe specific cell and/or tissue is inactivated.
 27. The chimericnon-human animal of claim 17, wherein the pluripotent cell is anembryonic stem cell.
 28. The chimeric non-human animal of claim 17,which is selected from the group consisting of mice, cattle, pigs,monkeys, rats, sheep, goats, rabbits, and hamsters.
 29. The chimericnon-human animal of claim 17, which is a mouse.
 30. The chimericnon-human animal of claim 17, wherein a combination of a gene that isexpressed in a specific cell and/or tissue and the cell and/or tissuedeficient in a non-human animal strain is selected from the groupconsisting of the following (1) to (7): (1) an immunoglobulin lightchain or heavy chain gene and a B-lymphocyte; (2) a T-cell receptor geneand a T-lymphocyte; (3) a myoglobin gene and a muscle cell; (4) acrystallin gene and a crystalline lens of an eyeball; (5) a renin geneand a kidney tissue; (6) an albumin gene and a liver tissue; and (7) alipase gene and a pancreas tissue.
 31. The chimeric non-human animal ofclaim 17, wherein the combination of a gene that is expressed in aspecific cell and/or tissue and the specific cell and/or tissuedeficient in a non-human animal strain is that of an immunoglobulinlight chain κ gene and a B-lymphocyte.
 32. A method for analyzing the invivo functions of a desired protein or a gene encoding the desiredprotein, which comprises comparing the phenotype of the chimericnon-human animal of any one of claims 17 to 31 or an offspring of thechimeric non-human animal capable of expressing the desired protein withthat of a corresponding wild-type non-human animal containing no nucleicacid sequence encoding the desired protein, so as to determinedifferences in these phenotypes.